{"id":8059,"date":"2023-05-13T11:39:13","date_gmt":"2023-05-13T04:09:13","guid":{"rendered":"https:\/\/abtvn.com\/?p=8059"},"modified":"2024-07-09T10:57:56","modified_gmt":"2024-07-09T03:27:56","slug":"real-time-pcr","status":"publish","type":"post","link":"https:\/\/abtvn.com\/en\/real-time-pcr\/","title":{"rendered":"T\u1ed5ng h\u1ee3p ki\u1ebfn th\u1ee9c chi ti\u1ebft v\u1ec1 k\u1ef9 thu\u1eadt Real-time PCR<\/strong>"},"content":{"rendered":"

1. Gi\u1edbi thi\u1ec7u v\u1ec1 k\u1ef9 thu\u1eadt real-time PCR<\/strong><\/h2>\n\n\n\n

Vi\u1ec7c ph\u00e1t minh ra ph\u1ea3n \u1ee9ng chu\u1ed7i polymerase (PCR)<\/a><\/strong> c\u1ee7a Kary Mullis v\u00e0o n\u0103m 1984 \u0111\u01b0\u1ee3c coi l\u00e0 m\u1ed9t cu\u1ed9c c\u00e1ch m\u1ea1ng trong khoa h\u1ecdc. Tuy nhi\u00ean, k\u1ebft qu\u1ea3 ph\u1ea3n \u1ee9ng PCR ch\u1ec9 \u0111\u01b0\u1ee3c bi\u1ebft sau khi ho\u00e0n th\u00e0nh v\u00e0 ph\u1ea3i th\u1ef1c hi\u1ec7n m\u1ed9t s\u1ed1 k\u1ef9 thu\u1eadt \u0111i k\u00e8m kh\u00e1c nh\u01b0 \u0111i\u1ec7n di hay \u0111o OD (Optical density). B\u00ean c\u1ea1nh \u0111\u00f3, k\u1ebft qu\u1ea3 ghi nh\u1eadn \u0111\u01b0\u1ee3c c\u1ee7a ph\u1ea3n \u1ee9ng l\u00e0 n\u1ed3ng \u0111\u1ed9 cu\u1ed1i c\u00f9ng c\u1ee7a s\u1ea3n ph\u1ea9m PCR, trong khi s\u1ef1 thay \u0111\u1ed5i n\u1ed3ng \u0111\u1ed9 trong qu\u00e1 tr\u00ecnh ph\u1ea3n \u1ee9ng c\u0169ng nh\u01b0 \u1ea3nh h\u01b0\u1edfng c\u1ee7a l\u01b0\u1ee3ng m\u1eabu \u0111\u1ea7u v\u00e0o \u0111\u1ebfn k\u1ebft qu\u1ea3 kh\u00f4ng \u0111\u01b0\u1ee3c ghi nh\u1eadn. <\/p>\n\n\n\n

\u0110\u1ec3 kh\u1eafc ph\u1ee5c \u0111\u01b0\u1ee3c nh\u1eefng v\u1ea5n \u0111\u1ec1 n\u00e0y, PCR \u0111\u00e3 \u0111\u01b0\u1ee3c c\u1ea3i ti\u1ebfn th\u00e0nh real-time PCR (hay PCR), \u0111\u00e2y \u0111\u01b0\u1ee3c coi l\u00e0 m\u1ed9t b\u01b0\u1edbc nh\u1ea3y v\u1ecdt v\u1ec1 c\u00f4ng ngh\u1ec7 v\u00e0o cu\u1ed1i th\u1ebf k\u1ef7 20, gi\u00fap m\u1edf ra nh\u1eefng \u1ee9ng d\u1ee5ng m\u1edbi v\u01b0\u1ee3t b\u1eadc cho c\u00e1c nh\u00e0 nghi\u00ean c\u1ee9u tr\u00ean to\u00e0n th\u1ebf gi\u1edbi. M\u00e1y real-time PCR l\u1ea7n \u0111\u1ea7u ti\u00ean \u0111\u01b0\u1ee3c th\u01b0\u01a1ng m\u1ea1i h\u00f3a b\u1edfi Applied Biosystems v\u00e0o n\u0103m 1996, c\u00f4ng ngh\u1ec7 n\u00e0y nhanh ch\u00f3ng ph\u00e1t tri\u1ec3n th\u00e0nh m\u1ed9t th\u1ecb tr\u01b0\u1eddng c\u1ea1nh tranh, h\u00e0ng lo\u1ea1t c\u00e1c c\u00f4ng ty kinh doanh s\u1ea3n ph\u1ea9m sinh h\u1ecdc cho ra m\u1eaft c\u00e1c d\u00f2ng m\u00e1y real-time PCR.<\/p>\n\n\n\n

V\u1ec1 m\u1eb7t \u0111\u1ecbnh ngh\u0129a, k\u1ef9 thu\u1eadt real-time PCR cho ph\u00e9p bi\u1ebft \u0111\u01b0\u1ee3c s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao DNA m\u1ee5c ti\u00eau t\u1ea1o ra ngay sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t c\u1ee7a ph\u1ea3n \u1ee9ng b\u1eb1ng c\u00f4ng ngh\u1ec7 hu\u1ef3nh quang. Ngo\u00e0i vi\u1ec7c c\u00f3 th\u1ec3 x\u00e1c \u0111\u1ecbnh \u0111\u01b0\u1ee3c s\u1ef1 c\u00f3 m\u1eb7t c\u1ee7a tr\u00ecnh t\u1ef1 DNA m\u1ee5c ti\u00eau (Real-time PCR \u0111\u1ecbnh t\u00ednh), qPCR c\u00f2n c\u00f3 th\u1ec3 \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u01b0\u1ee3c DNA m\u1ee5c ti\u00eau (qPCR \u0111\u1ecbnh l\u01b0\u1ee3ng) c\u00f3 trong m\u1eabu ban \u0111\u1ea7u qua m\u1ed7i chu k\u1ef3.<\/p>\n\n\n\n

2. Nguy\u00ean l\u00fd ho\u1ea1t \u0111\u1ed9ng c\u1ee7a k\u1ef9 thu\u1eadt real-time PCR<\/strong><\/h2>\n\n\n\n

T\u01b0\u01a1ng t\u1ef1 nh\u01b0 PCR, k\u1ef9 thu\u1eadt real-time PCR c\u0169ng bao g\u1ed3m c\u00e1c th\u00e0nh ph\u1ea7n c\u01a1 b\u1ea3n nh\u01b0 dNTP, DNA Polymerase, DNA m\u1ea1ch khu\u00f4n, c\u1eb7p m\u1ed3i v\u00e0 dung d\u1ecbch \u0111\u1ec7m. \u0110i\u1ec3m kh\u00e1c bi\u1ec7t \u0111\u00f3 l\u00e0 real-time PCR s\u1eed d\u1ee5ng ch\u1ea5t ph\u00e1t hu\u1ef3nh quang \u0111\u1ec3 m\u00e1y c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n v\u00e0 \u0111o \u0111\u01b0\u1ee3c c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u t\u1eeb ch\u1ea5t n\u00e0y. Khi ph\u1ea3n \u1ee9ng nh\u00e2n b\u1ea3n x\u1ea3y ra t\u1edbi m\u1ed9t chu k\u1ef3 nh\u1ea5t \u0111\u1ecbnh, c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang s\u1ebd b\u1eaft \u0111\u1ea7u c\u00f3 s\u1ef1 gia t\u0103ng r\u00f5 r\u1ec7t v\u00e0 t\u01b0\u01a1ng quan v\u1edbi s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n sao DNA \u0111\u01b0\u1ee3c t\u1ea1o ra. <\/p>\n\n\n\n

K\u1ebft qu\u1ea3 \u0111\u01b0\u1ee3c th\u1ec3 hi\u1ec7n d\u01b0\u1edbi d\u1ea1ng bi\u1ec3u \u0111\u1ed3 quan s\u00e1t \u0111\u01b0\u1ee3c qua m\u1ed7i chu k\u1ef3, t\u1eeb \u0111\u00f3 c\u00f3 th\u1ec3 \u0111\u01b0a ra \u0111\u00e1nh gi\u00e1 v\u1ec1 hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i DNA m\u1ee5c ti\u00eau. Real-time PCR y\u00eau c\u1ea7u c\u00f3 thi\u1ebft b\u1ecb \u0111o c\u01b0\u1eddng \u0111\u1ed9 ph\u00e1t hu\u1ef3nh quang t\u1eeb \u1ed1ng m\u1eabu v\u00e0 c\u00e0i ch\u01b0\u01a1ng tr\u00ecnh ph\u1ea7n m\u1ec1m cho ph\u00e9p x\u1eed l\u00fd k\u1ebft qu\u1ea3 v\u1ec1 s\u1ef1 bi\u1ebfn \u0111\u1ed5i c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang.<\/p>\n\n\n\n

Chu tr\u00ecnh nhi\u1ec7t c\u1ee7a real-time PCR c\u0169ng c\u00f3 3 giai \u0111o\u1ea1n c\u01a1 b\u1ea3n t\u01b0\u01a1ng t\u1ef1 nh\u01b0 PCR bao g\u1ed3m:<\/p>\n\n\n\n

– Giai \u0111o\u1ea1n bi\u1ebfn t\u00ednh: H\u1ed7n h\u1ee3p ph\u1ea3n \u1ee9ng \u0111\u01b0\u1ee3c gia nhi\u1ec7t \u0111\u1ebfn 94-950<\/sup>C, l\u00fac n\u00e0y li\u00ean k\u1ebft hydro gi\u1eefa c\u00e1c baz\u01a1 trong s\u1ee3i DNA m\u1ea1ch \u0111\u00f4i b\u1ecb ph\u00e1 v\u1ee1, d\u1eabn \u0111\u1ebfn t\u1ea1o th\u00e0nh 2 s\u1ee3i \u0111\u01a1n DNA. M\u1ed7i s\u1ee3i \u0111\u01a1n n\u00e0y tr\u1edf th\u00e0nh khu\u00f4n \u0111\u1ec3 t\u1ed5ng h\u1ee3p m\u1ea1ch m\u1edbi. Th\u1eddi gian bi\u1ebfn t\u00ednh c\u00f3 th\u1ec3 t\u0103ng l\u00ean n\u1ebfu th\u00e0nh ph\u1ea7n khu\u00f4n c\u00f3 nhi\u1ec1u GC.<\/p>\n\n\n\n

– Giai \u0111o\u1ea1n b\u1eaft c\u1eb7p: Nhi\u1ec7t \u0111\u1ed9 ph\u1ea3n \u1ee9ng gi\u1ea3m xu\u1ed1ng c\u00f2n 50-650<\/sup>C trong 20-40 gi\u00e2y \u0111\u1ec3 probe v\u00e0 m\u1ed3i g\u1eafn l\u1ea7n l\u01b0\u1ee3t v\u00e0o s\u1ee3i DNA khu\u00f4n v\u00e0 b\u1eaft \u0111\u1ea7u qu\u00e1 tr\u00ecnh t\u1ed5ng h\u1ee3p m\u1ea1ch m\u1edbi nh\u1edd v\u00e0o s\u1ef1 ho\u1ea1t \u0111\u1ed9ng c\u1ee7a DNA polymerase.<\/p>\n\n\n\n

– Giai \u0111o\u1ea1n k\u00e9o d\u00e0i: Nhi\u1ec7t \u0111\u1ed9 ph\u1ea3n \u1ee9ng t\u0103ng l\u00ean 720<\/sup>C, \u0111\u00e2y l\u00e0 nhi\u1ec7t \u0111\u1ed9 t\u1ed1i \u01b0u cho ho\u1ea1t \u0111\u1ed9ng t\u1ed5ng h\u1ee3p m\u1ea1ch m\u1edbi c\u1ee7a DNA polymerase. Giai \u0111o\u1ea1n n\u00e0y kh\u00f4ng b\u1eaft bu\u1ed9c \u1edf m\u1ed9t s\u1ed1 chu tr\u00ecnh qPCR, do k\u1ef9 thu\u1eadt n\u00e0y th\u01b0\u1eddng d\u00f9ng khu\u1ebfch \u0111\u1ea1i c\u00e1c \u0111o\u1ea1n c\u00f3 tr\u00ecnh t\u1ef1 ng\u1eafn h\u01a1n PCR, v\u00e0o kho\u1ea3ng <200bp, do v\u1eady ch\u1ec9 c\u1ea7n hai b\u01b0\u1edbc bi\u1ebfn t\u00ednh v\u00e0 b\u1eaft c\u1eb7p l\u00e0 \u0111\u1ee7 \u0111\u1ec3 khu\u1ebfch \u0111\u1ea1i \u0111o\u1ea1n gen m\u1ee5c ti\u00eau trong ph\u1ea3n \u1ee9ng qPCR. Chu tr\u00ecnh ch\u1ec9 g\u1ed3m 2 b\u01b0\u1edbc gi\u00fap ti\u1ebft ki\u1ec7m th\u1eddi gian, nhanh ch\u00f3ng ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u01b0\u1ee3c DNA.<\/p>\n\n\n\n

C\u00e1c giai \u0111o\u1ea1n n\u00e0y \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n th\u00f4ng qua s\u1ef1 lu\u00e2n chuy\u1ec3n nhi\u1ec7t \u0111\u1ed9 gi\u1eefa c\u00e1c chu k\u1ef3. Qua m\u1ed7i chu k\u1ef3, t\u00edn hi\u1ec7u hu\u1ef3nh quang s\u1ebd \u0111\u01b0\u1ee3c ghi nh\u1eadn c\u00f3 s\u1ef1 gia t\u0103ng t\u01b0\u01a1ng \u1ee9ng v\u1edbi l\u01b0\u1ee3ng b\u1ea3n sao DNA nh\u00e2n l\u00ean theo c\u1ea5p s\u1ed1 l\u0169y th\u1eeba.<\/p>\n\n\n\n

3. Th\u00e0nh ph\u1ea7n c\u1ee7a ph\u1ea3n \u1ee9ng real-time PCR<\/strong><\/h2>\n\n\n\n

Khi th\u1ef1c hi\u1ec7n real-time PCR, c\u00e1c th\u00e0nh ph\u1ea7n ph\u1ea3n \u1ee9ng s\u1ebd \u0111\u01b0\u1ee3c tr\u1ed9n v\u1edbi nhau trong tube v\u00e0 \u0111\u1eb7t trong m\u00e1y real-time PCR \u0111\u1ec3 th\u1ef1c hi\u1ec7n qu\u00e1 tr\u00ecnh nh\u00e2n b\u1ea3n DNA. Th\u00e0nh ph\u1ea7n ph\u1ea3n \u1ee9ng qPCR bao g\u1ed3m: m\u1eabu (khu\u00f4n DNA\/RNA), m\u1ed3i, ch\u1ea5t hu\u1ef3nh quang, \u0111\u1ec7m, DNA polymerase, nucleotide t\u1ef1 do (dNTP) v\u00e0 c\u00e1c ch\u1ea5t h\u1ed7 tr\u1ee3 (n\u1ebfu c\u1ea7n). Hi\u1ec7n nay c\u00f3 hai c\u00e1ch th\u1ee9c \u0111\u1ec3 chu\u1ea9n b\u1ecb cho ph\u1ea3n \u1ee9ng qPCR, \u0111\u00f3 l\u00e0 ph\u1ed1i tr\u1ed9n c\u00e1c th\u00e0nh ph\u1ea7n \u0111\u01a1n l\u1ebb ho\u1eb7c s\u1eed d\u1ee5ng qPCR mix (c\u00e1c th\u00e0nh ph\u1ea7n \u0111\u01b0\u1ee3c ph\u1ed1i tr\u1ed9n s\u1eb5n th\u00e0nh m\u1ed9t lo\u1ea1i dung d\u1ecbch)<\/p>\n\n\n\n

3.1. M\u1eabu (khu\u00f4n DNA\/RNA)<\/strong><\/h3>\n\n\n\n

Th\u00f4ng th\u01b0\u1eddng, m\u1eabu tr\u01b0\u1edbc khi \u0111\u01b0a v\u00e0o ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i c\u1ea7n \u0111\u01b0\u1ee3c x\u1eed l\u00fd m\u1eabu v\u00e0 t\u00e1ch chi\u1ebft DNA\/RNA<\/a><\/strong>. Vi\u1ec7c l\u1ea5y m\u1eabu c\u1ea7n \u0111\u1ea3m b\u1ea3o l\u1ea5y \u0111\u00fang v\u1ecb tr\u00ed, l\u1ea5y \u0111\u1ee7 l\u01b0\u1ee3ng m\u1eabu c\u1ea7n thi\u1ebft \u0111\u1ec3 \u0111\u1ea3m b\u1ea3o k\u1ebft qu\u1ea3 c\u1ee7a x\u00e9t nghi\u1ec7m qPCR. Ngu\u1ed3n m\u1eabu hi\u1ec7n nay r\u1ea5t \u0111a d\u1ea1ng nh\u01b0 m\u1eabu m\u00e1u, m\u1eabu t\u1ebf b\u00e0o nu\u00f4i c\u1ea5y, vi khu\u1ea9n, huy\u1ec5n d\u1ecbch (huy\u1ec1n ph\u00f9), m\u1eabu qu\u00e9t b\u1ec1 m\u1eb7t, m\u1eabu d\u1ecbch ph\u1ebft (y t\u1ebf), \u2026 m\u1ed7i lo\u1ea1i m\u1eabu s\u1ebd c\u00f3 c\u00e1ch x\u1eed l\u00fd kh\u00e1c nhau \u0111\u1ec3 \u0111\u1ea3m b\u1ea3o m\u1eabu \u0111\u1ea7u v\u00e0o cho x\u00e9t nghi\u1ec7m PCR.<\/p>\n\n\n\n

Sau khi x\u1eed l\u00fd m\u1eabu, b\u01b0\u1edbc t\u00e1ch chi\u1ebft DNA\/RNA c\u1ea7n \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n \u0111\u1ec3 thu nh\u1eadn ngu\u1ed3n m\u1eabu \u0111\u1ea7u v\u00e0o ch\u1ea5t l\u01b0\u1ee3ng cho PCR. Hi\u1ec7n nay, c\u00f3 nhi\u1ec1u k\u1ef9 thu\u1eadt t\u00e1ch chi\u1ebft DNA\/RNA \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng nh\u01b0 ph\u1ed5 bi\u1ebfn, bao g\u1ed3m:<\/p>\n\n\n\n

 – T\u00e1ch th\u00f4: ph\u01b0\u01a1ng ph\u00e1p n\u00e0y ph\u00e1 v\u1ee1 t\u1ebf b\u00e0o, gi\u1ea3i ph\u00f3ng nucleic acid, kh\u00f4ng c\u00f3 b\u01b0\u1edbc ph\u00e2n l\u1eadp ho\u00e0n to\u00e0n nucleic acid ra kh\u1ecfi t\u1ea1p ch\u1ea5t.<\/p>\n\n\n\n

– Ph\u01b0\u01a1ng ph\u00e1p t\u1ee7a: s\u1eed d\u1ee5ng ph\u01b0\u01a1ng ph\u00e1p t\u1ee7a DNA\/RNA b\u1eb1ng h\u00f3a ch\u1ea5t v\u00e0 l\u1ef1c ly t\u00e2m \u0111\u1ec3 ph\u00e2n t\u00e1ch ph\u1ea7n l\u1edbn nucleic acid ra kh\u1ecfi t\u1ea1p ch\u1ea5t. M\u1ed9t s\u1ed1 h\u00f3a ch\u1ea5t th\u01b0\u1eddng s\u1eed d\u1ee5ng l\u00e0 phenol\/chloform, mu\u1ed1i hay CTAB.<\/p>\n\n\n\n

– Ph\u01b0\u01a1ng ph\u00e1p d\u1ef1a tr\u00ean l\u1ef1c li\u00ean k\u1ebft gi\u1eefa DNA v\u1edbi ph\u00e2n t\u1eed silica: s\u1eed d\u1ee5ng l\u1ef1c li\u00ean k\u1ebft gi\u1eefa nucleic acid v\u00e0 h\u1ea1t silica \u0111\u1ec3 ph\u00e2n l\u1eadp nucleic acid ra kh\u1ecfi t\u1ea1p ch\u1ea5t. Bao g\u1ed3m ph\u01b0\u01a1ng ph\u00e1p s\u1eed d\u1ee5ng c\u1ed9t silica (t\u00e1ch c\u1ed9t), h\u1ea1t silica t\u1ef1 do (ph\u01b0\u01a1ng ph\u00e1p Boom) v\u00e0 h\u1ea1t t\u1eeb bao ph\u1ee7 silica (t\u00e1ch m\u00e1y t\u1ef1 \u0111\u1ed9ng).<\/p>\n\n\n\n

Trong \u0111\u00f3 ph\u01b0\u01a1ng ph\u00e1p t\u00e1ch c\u1ed9t v\u00e0 h\u1ea1t t\u1eeb bao ph\u1ee7 silica g\u1ea7n \u0111\u00e2y \u0111\u00e3 \u0111\u01b0\u1ee3c th\u01b0\u01a1ng m\u1ea1i h\u00f3a th\u00e0nh c\u00e1c b\u1ed9 s\u1ea3n ph\u1ea9m d\u00f9ng cho t\u00e1ch chi\u1ebft DNA\/RNA. C\u00e1c b\u1ed9 kit t\u00e1ch chi\u1ebft hi\u1ec7n \u0111ang l\u00e0 gi\u1ea3i ph\u00e1p \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng ph\u1ed5 bi\u1ebfn v\u1edbi quy tr\u00ecnh th\u1ef1c hi\u1ec7n \u0111\u01a1n gi\u1ea3n, nhanh ch\u00f3ng v\u00e0 ti\u1ec7n l\u1ee3i. C\u00e1c b\u1ed9 kit th\u01b0\u1eddng kh\u00e1c nhau \u1edf dung d\u1ecbch \u0111\u1ec7m ly gi\u1ea3i t\u01b0\u01a1ng \u1ee9ng v\u1edbi m\u1ee5c \u0111\u00edch t\u00e1ch chi\u1ebft t\u1eebng lo\u1ea1i m\u1eabu kh\u00e1c nhau, gi\u00fap r\u00fat ng\u1eafn th\u1eddi gian t\u00e1ch chi\u1ebft, hi\u1ec7u su\u1ea5t thu h\u1ed3i v\u00e0 \u0111\u1ed9 tinh s\u1ea1ch DNA\/RNA cao.<\/p>\n\n\n\n

3.2. M\u1ed3i (Primer)<\/strong><\/h3>\n\n\n\n

M\u1ed3i bao g\u1ed3m m\u1ed3i xu\u00f4i (forward primer) v\u00e0 m\u1ed3i ng\u01b0\u1ee3c (revverse primer), l\u00e0 nh\u1eefng \u0111o\u1ea1n oligonucleotide ng\u1eafn (20-30 bp) c\u00f3 tr\u00ecnh t\u1ef1 \u1edf \u0111\u1ea7u 5\u2019 b\u1eaft c\u1eb7p \u0111\u1eb7c hi\u1ec7u v\u1edbi \u0111\u1ea7u 3\u2019 c\u1ee7a DNA m\u1ee5c ti\u00eau. \u1ede giai \u0111o\u1ea1n b\u1eaft c\u1eb7p, c\u1eb7p m\u1ed3i s\u1ebd g\u1eafn t\u01b0\u01a1ng \u1ee9ng l\u00ean hai s\u1ee3i \u0111\u01a1n c\u1ee7a DNA sau khi b\u1ecb bi\u1ebfn t\u00ednh v\u00e0 khu\u1ebfch \u0111\u1ea1i \u0111o\u1ea1n tr\u00ecnh t\u1ef1 n\u1eb1m gi\u1eefa hai m\u1ed3i. V\u1ecb tr\u00ed m\u1ed3i g\u1eafn v\u00e0o gen m\u1ee5c ti\u00eau c\u0169ng l\u00e0 \u0111i\u1ec3m kh\u1edfi \u0111\u1ea7u \u0111\u1ec3 c\u00e1c DNA polymerase g\u1eafn v\u00e0o v\u00e0 c\u00f3 th\u1ec3 th\u1ef1c hi\u1ec3n t\u1ed5ng h\u1ee3p m\u1ea1ch b\u1ed5 sung m\u1edbi. <\/p>\n\n\n\n

Do \u0111\u00f3, m\u1ed3i l\u00e0 ch\u1ec9 ti\u00eau quan tr\u1ecdng quy\u1ebft \u0111\u1ecbnh m\u1ee9c \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u v\u00e0 n\u0103ng su\u1ea5t c\u1ee7a ph\u1ea3n \u1ee9ng PCR n\u00ean c\u1ea7n \u0111\u00e1p \u1ee9ng c\u00e1c y\u00eau c\u1ea7u sau:<\/p>\n\n\n\n

– V\u1ec1 tr\u00ecnh t\u1ef1, c\u1ea3 hai m\u1ed3i \u0111\u1ec1u ph\u1ea3i \u0111\u1eb7c tr\u01b0ng cho khu\u00f4n DNA m\u1ee5c ti\u00eau, h\u1ea1n ch\u1ebf t\u1ed1i thi\u1ec3u tr\u00f9ng v\u1edbi c\u00e1c gen kh\u00e1c. M\u1ed3i xu\u00f4i v\u00e0 m\u1ed3i ng\u01b0\u1ee3c kh\u00f4ng c\u00f3 s\u1ef1 b\u1eaft c\u1eb7p b\u1ed5 sung (cross dimer), kh\u00f4ng t\u1ea1o c\u00e1c c\u1ea5u tr\u00fac th\u1ee9 c\u1ea5p kh\u00e1c nh\u01b0 t\u1ef1 b\u1eaft c\u1eb7p (self dimer) hay c\u1ea5u tr\u00fac \u201ck\u1eb9p t\u00f3c\u201d (hairpin). <\/p>\n\n\n\n

B\u00ean c\u1ea1nh \u0111\u00f3, th\u00e0nh ph\u1ea7n GC c\u0169ng n\u00ean \u0111\u01b0\u1ee3c c\u00e2n \u0111\u1ed1i \u0111\u1ec3 \u0111\u1ea3m b\u1ea3o s\u1ef1 b\u1eaft c\u1eb7p, vi\u1ec7c c\u00f3 m\u1eb7t GC s\u1ebd gi\u00fap m\u1ed3i b\u00e1m v\u00e0 k\u00e9o d\u00e0i (n\u00ean c\u00f3 1-2 G ho\u1eb7c C \u1edf \u0111\u1ea7u 3\u2019), nh\u01b0ng n\u1ebfu c\u00f3 nhi\u1ec1u GC l\u1eb7p \u0111i l\u1eb7p l\u1ea1i s\u1ebd l\u00e0m m\u1ed3i b\u1eaft c\u1eb7p kh\u00f4ng \u0111\u1eb7c hi\u1ec7u (th\u01b0\u1eddng t\u1eeb 3 G ho\u1eb7c C \u1edf \u0111\u1ea7u 3\u2019). H\u00e0m l\u01b0\u1ee3ng GC ph\u00f9 h\u1ee3p l\u00e0 kho\u1ea3ng t\u1eeb 40-60 % \u0111\u1ec3 tr\u00e1nh s\u1ef1 b\u1eaft c\u1eb7p m\u1ed3i kh\u00f4ng \u0111\u00fang.<\/p>\n\n\n\n

– V\u1ec1 nhi\u1ec7t \u0111\u1ed9 b\u1eaft c\u1eb7p, m\u1ed3i c\u00f3 th\u1ec3 b\u00e1m t\u1ed1t l\u00ean DNA khu\u00f4n khi \u1edf nhi\u1ec7t \u0111\u1ed9 ph\u00f9 h\u1ee3p, g\u1ecdi l\u00e0 nhi\u1ec7t \u0111\u1ed9 b\u1eaft c\u1eb7p (annealing Temperature-Ta<\/sub>), l\u00fac n\u00e0y nhi\u1ec7t \u0111\u1ed9 gi\u1ea3m xu\u1ed1ng c\u00f2n 50-65\u00b0C trong 20-40 gi\u00e2y \u0111\u1ec3 hai m\u1ed3i g\u1eafn v\u00e0o s\u1ee3i DNA. Nhi\u1ec7t \u0111\u1ed9 n\u00e0y c\u1ea7n ph\u1ea3i \u0111\u1ee7 th\u1ea5p \u0111\u1ec3 cho ph\u00e9p m\u1ed3i b\u1eaft c\u1eb7p v\u1edbi khu\u00f4n, nh\u01b0ng c\u0169ng ph\u1ea3i \u0111\u1ee7 cao cho qu\u00e1 tr\u00ecnh b\u1eaft c\u1eb7p \u0111\u1eb7c hi\u1ec7u. Tr\u01b0\u1eddng h\u1ee3p nhi\u1ec7t \u0111\u1ed9 Ta qu\u00e1 th\u1ea5p th\u00ec s\u1ea3n ph\u1ea9m kh\u00f4ng \u0111\u1eb7c hi\u1ec7u s\u1ebd \u0111\u01b0\u1ee3c t\u1ea1o ra nhi\u1ec1u, trong khi qu\u00e1 cao c\u00f3 th\u1ec3 d\u1eabn \u0111\u1ebfn l\u01b0\u1ee3ng s\u1ea3n ph\u1ea9m PCR kh\u00f4ng \u0111\u01b0\u1ee3c t\u1ea1o ra. <\/p>\n\n\n\n

Aa<\/sub> \u0111\u01b0\u1ee3c t\u00ednh d\u1ef1a tr\u00ean nhi\u1ec7t \u0111\u1ed9 n\u00f3ng ch\u1ea3y c\u1ee7a m\u1ed3i (melting Temperature-Tm<\/sub>). Tm<\/sub> \u0111\u01b0\u1ee3c \u0111\u1ecbnh ngh\u0129a l\u00e0 \u0111i\u1ec3m nhi\u1ec7t m\u00e0 t\u1ea1i \u0111\u00f3 50% s\u1ee3i \u0111\u00f4i DNA ph\u00e2n t\u00e1ch nhau v\u00e0 t\u1ea1o s\u1ee3i \u0111\u01a1n, Ta<\/sub> th\u01b0\u1eddng th\u1ea5p h\u01a1n Tm<\/sub> kho\u1ea3ng 3-5 o<\/sup>C. Nhi\u1ec7t \u0111\u1ed9 Tm<\/sub> c\u1ee7a hai m\u1ed3i kh\u00f4ng ch\u00eanh l\u1ec7ch nhau qu\u00e1 2o<\/sup>C, nhi\u1ec7t \u0111\u1ed9 ch\u00eanh l\u1ec7ch qu\u00e1 5o<\/sup>C c\u00f3 th\u1ec3 kh\u00f4ng th\u1ec3 t\u1ea1o ra \u0111\u01b0\u1ee3c s\u1ea3n ph\u1ea9m. C\u00f4ng th\u1ee9c t\u00ednh Tm<\/sub> nh\u01b0 sau: Tm<\/sub>=4\u00b0C x (G + C) + 2\u00b0C x (A + T). T\u1eeb c\u00f4ng th\u1ee9c t\u00ednh Tm<\/sub>, Ta<\/sub> \u0111\u01b0\u1ee3c t\u00ednh nh\u01b0 sau: Ta<\/sub> = 0.3 x Tm <\/sub>(M\u1ed3i) + 0.7 Tm<\/sub> (s\u1ea3n ph\u1ea9m) \u2013 14.9<\/p>\n\n\n\n

– V\u1ec1 n\u1ed3ng \u0111\u1ed9, m\u1ed3i th\u01b0\u1eddng \u0111\u01b0\u1ee3c cho v\u00e0o ph\u1ea3n \u1ee9ng PCR dao \u0111\u1ed9ng t\u1eeb 0,1-1M. Vi\u1ec7c s\u1eed d\u1ee5ng m\u1ed3i th\u00f4ng th\u01b0\u1eddng d\u1ef1a tr\u00ean khuy\u1ebfn c\u00e1o c\u1ee7a nh\u00e0 s\u1ea3n xu\u1ea5t hay h\u01b0\u1edbng d\u1eabn trong b\u1ed9 kit PCR g\u1ecdi l\u00e0 n\u1ed3ng \u0111\u1ed9 m\u1ed3i ti\u00eau chu\u1ea9n v\u00e0 ch\u1ec9 n\u00ean thay \u0111\u1ed5i khi c\u1ea7n. N\u1ebfu n\u1ed3ng \u0111\u1ed9 m\u1ed3i qu\u00e1 th\u1ea5p th\u00ec s\u1ebd kh\u00f4ng \u0111\u1ea3m b\u1ea3o l\u01b0\u1ee3ng m\u1ed3i \u0111\u1ee7 \u0111\u1ec3 ho\u00e0n th\u00e0nh chu tr\u00ecnh PCR, ng\u01b0\u1ee3c l\u1ea1i n\u1ebfu n\u1ed3ng \u0111\u1ed9 qu\u00e1 cao th\u00ec d\u1ec5 g\u00e2y vi\u1ec7c khu\u1ebfch \u0111\u1ea1i s\u1ea3n ph\u1ea9m kh\u00f4ng \u0111\u1eb7c hi\u1ec7u.<\/p>\n\n\n\n

Vi\u1ec7c thi\u1ebft k\u1ebf m\u1ed3i c\u00f3 th\u1ec3 l\u00e0m th\u1ee7 c\u00f4ng d\u1ef1a tr\u00ean c\u00e1c nguy\u00ean t\u1eafc trong thi\u1ebft k\u1ebf m\u1ed3i, tuy nhi\u00ean c\u00e1ch n\u00e0y th\u01b0\u1eddng s\u1ebd g\u1eb7p kh\u00f3 kh\u0103n n\u1ebfu c\u00f3 s\u1ed1 l\u01b0\u1ee3ng DNA khu\u00f4n nhi\u1ec1u, c\u00f3 r\u1ea5t nhi\u1ec1u th\u00f4ng s\u1ed1 ph\u1ea3i t\u00ednh to\u00e1n. Do v\u1eady, hi\u1ec7n nay \u0111\u00e3 c\u00f3 th\u00eam m\u1ed9t s\u1ed1 ph\u1ea7n m\u1ec1m, c\u00f4ng c\u1ee5 tr\u1ef1c tuy\u1ebfn h\u1ed7 tr\u1ee3 hay c\u00f3 th\u1ec3 s\u1eed d\u1ee5ng tr\u1ef1c ti\u1ebfp \u0111\u1ec3 thi\u1ebft k\u1ebf m\u1ed3i d\u1ef1a tr\u00ean tr\u00ecnh t\u1ef1 DNA khu\u00f4n m\u1eabu. C\u00e1c c\u00f4ng c\u1ee5 n\u00e0y gi\u00fap ng\u01b0\u1eddi d\u00f9ng ch\u1ecdn \u0111\u01b0\u1ee3c tr\u00ecnh t\u1ef1 c\u1eb7p m\u1ed3i t\u1ed1i \u01b0u d\u1ef1a tr\u00ean c\u00e1c tham s\u1ed1 x\u00e1c \u0111\u1ecbnh. M\u1ed9t s\u1ed1 ph\u1ea7n m\u1ec1m, c\u00f4ng c\u1ee5 thi\u1ebft k\u1ebf m\u1ed3i ph\u1ed5 bi\u1ebfn l\u00e0 Primer 3, Primer-BLAST, FastPCR, \u2026<\/p>\n\n\n\n

3.3. Enzyme t\u1ed5ng h\u1ee3p (DNA polymerase)<\/strong><\/h3>\n\n\n\n

DNA polymerase l\u00e0 th\u00e0nh ph\u1ea7n quan tr\u1ecdng trong ph\u1ea3n \u1ee9ng nh\u1edd ho\u1ea1t t\u00ednh t\u1ed5ng h\u1ee3p d\u1ea1ng chu\u1ed7i nh\u1eb1m m\u1ee5c \u0111\u00edch khu\u1ebfch \u0111\u1ea1i \u0111\u01b0\u1ee3c tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau. Hi\u1ec7n nay, Taq<\/em> polymerase v\u1edbi ho\u1ea1t t\u00ednh ch\u1ecbu nhi\u1ec7t k\u1ebft h\u1ee3p v\u1edbi c\u00f4ng ngh\u1ec7 kh\u1edfi \u0111\u1ed9ng b\u1eb1ng gia nhi\u1ec7t (Hot-start) \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng ph\u1ed5 bi\u1ebfn. Tuy nhi\u00ean, nh\u01b0\u1ee3c \u0111i\u1ec3m c\u1ee7a Taq<\/em> l\u00e0 kh\u00f4ng c\u00f3 ho\u1ea1t t\u00ednh s\u1eeda sai exonuclease 3\u2019-5\u2019, do v\u1eady c\u00f3 th\u1ec3 d\u1eabn \u0111\u1ebfn x\u1ea3y ra khu\u1ebfch \u0111\u1ea1i tr\u00ecnh t\u1ef1 sai. V\u00ec v\u1eady, nh\u1eb1m kh\u1eafc ph\u1ee5c h\u1ea1n ch\u1ebf c\u1ee7a Taq<\/em>, m\u1ed9t s\u1ed1 polymerase \u0111\u01b0\u1ee3c c\u1ea3i ti\u1ebfn nh\u01b0 Pfu<\/em> v\u1edbi c\u01a1 ch\u1ebf s\u1eeda sai n\u00ean c\u00f3 th\u1ec3 l\u00e0m gi\u1ea3m m\u1ed9t c\u00e1ch \u0111\u00e1ng k\u1ec3 s\u1ed1 \u0111\u1ed9t bi\u1ebfn x\u1ea3y ra khi khu\u1ebfch \u0111\u1ea1i. <\/p>\n\n\n\n

Ng\u00e0y nay, m\u1ed9t s\u1ed1 polymerase v\u1edbi hi\u1ec7u su\u1ea5t cao c\u00f3 th\u1ec3 cung c\u1ea5p c\u1ea3 \u0111\u1ed9 tin c\u1eady cao l\u1eabn s\u1ef1 nh\u00e2n b\u1ea3n ch\u00ednh x\u00e1c c\u1ee7a DNA gi\u00fap gia t\u0103ng hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i trong t\u1ea1o d\u00f2ng, gen m\u1ee5c ti\u00eau d\u00e0i hay gi\u00e0u CG. Trong ph\u1ea3n \u1ee9ng PCR, polymerase th\u00f4ng th\u01b0\u1eddng \u0111\u01b0\u1ee3c d\u00f9ng kho\u1ea3ng 1-2 \u0111\u01a1n v\u1ecb IU (international unit) trong th\u1ec3 t\u00edch t\u1ed5ng l\u00e0 50 \u00b5L, n\u1ebfu m\u1eabu c\u00f3 ch\u1ea5t \u1ee9c ch\u1ebf th\u00ec c\u00f3 th\u1ec3 t\u0103ng th\u00eam l\u01b0\u1ee3ng enzyme nh\u01b0ng n\u1ebfu t\u0103ng qu\u00e1 cao th\u00ec c\u00f3 th\u1ec3 t\u1ea1o s\u1ea3n ph\u1ea9m kh\u00f4ng \u0111\u1eb7c hi\u1ec7u.<\/p>\n\n\n\n

3.4. Deoxynucleotide triphosphate (dNTP)<\/strong><\/h3>\n\n\n\n

Deoxynucleotide triphosphate (dNTP) l\u00e0 nh\u1eefng \u0111\u01a1n v\u1ecb nucleotide ri\u00eang l\u1ebb d\u00f9ng \u0111\u1ec3 t\u1ed5ng h\u1ee3p m\u1ea1ch m\u1edbi trong ph\u1ea3n \u1ee9ng qPCR. dNTP bao g\u1ed3m 4 lo\u1ea1i l\u00e0 adenine (dATP), cytosine (dCTP), guanine (dGTP) v\u00e0 thymine (dTTP). C\u1ea5u t\u1ea1o c\u1ee7a dNTP \u0111\u1ec1u bao g\u1ed3m m\u1ed9t lo\u1ea1i base \u1edf carbone v\u1ecb tr\u00ed s\u1ed1 1 (C1) v\u00e0 3 ph\u00e2n t\u1eed phosphate (triphosphate) \u0111\u01b0\u1ee3c g\u1eafn t\u1ea1i carbon s\u1ed1 5 (C5) v\u00e0 \u0111\u00e2y c\u0169ng v\u1ecb tr\u00ed dNTP g\u1eafn v\u00e0o \u0111\u1ea7u 3\u2019 c\u1ee7a s\u1ee3i b\u1ed5 sung tr\u00ean DNA khu\u00f4n. N\u0103ng l\u01b0\u1ee3ng \u0111\u1ec3 ph\u1ea3n \u1ee9ng n\u00e0y x\u1ea3y ra \u0111\u01b0\u1ee3c l\u1ea5y t\u1eeb c\u00e1c li\u00ean k\u1ebft phosphate gi\u00e0u n\u0103ng l\u01b0\u1ee3ng c\u1ee7a nh\u00f3m triphosphate.<\/p>\n\n\n\n

Hi\u1ec7n nay, dNTP c\u00f3 th\u1ec3 \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng theo nhi\u1ec1u c\u00e1ch, m\u1ed9t l\u00e0 t\u1eebng lo\u1ea1i dNTP s\u1ebd \u0111\u01b0\u1ee3c b\u01a1m ri\u00eang l\u1ebb v\u00e0o \u1ed1ng ph\u1ea3n \u1ee9ng qPCR, hai l\u00e0 b\u1ed1n lo\u1ea1i dNTP \u0111\u01b0\u1ee3c ph\u1ed1i tr\u1ed9n l\u1ea1i th\u00e0nh d\u1ea1ng mix (dNTP mix) tr\u01b0\u1edbc \u0111\u00f3 v\u00e0 cu\u1ed1i c\u00f9ng dNTP l\u00e0 th\u00e0nh ph\u1ea7n s\u1eb5n c\u00f3 trong qPCR mix bao g\u1ed3m nh\u1eefng th\u00e0nh ph\u1ea7n kh\u00e1c m\u00e0 ng\u01b0\u1eddi d\u00f9ng ch\u1ec9 c\u1ea7n b\u1ed5 sung th\u00eam DNA khu\u00f4n v\u00e0 n\u01b0\u1edbc l\u00e0 c\u00f3 th\u1ec3 ch\u1ea1y ph\u1ea3n \u1ee9ng qPCR. <\/p>\n\n\n\n

C\u00e1c dNTP hi\u1ec7n nay \u0111\u01b0\u1ee3c tinh ch\u1ebf b\u1eb1ng ph\u01b0\u01a1ng ph\u00e1p s\u1eafc k\u00fd hi\u1ec7u n\u0103ng l\u1ecfng (HPLC) v\u00e0 c\u00f3 \u0111\u1ed9 tinh khi\u1ebft \u00edt nh\u1ea5t l\u00e0 99,5%. B\u1ed1n lo\u1ea1i dNTP \u0111\u1ec1u \u0111\u01b0\u1ee3c d\u00f9ng v\u1edbi n\u1ed3ng \u0111\u1ed9 gi\u1ed1ng nhau khi ph\u1ed1i tr\u1ed9n c\u00f9ng nhau hay d\u00f9ng ri\u00eang l\u1ebb, n\u1ed3ng \u0111\u1ed9 cu\u1ed1i dao \u0111\u1ed9ng trong kho\u1ea3ng 20-200 \u00b5M. Trong tr\u01b0\u1eddng h\u1ee3p g\u00e2y \u0111\u1ed9t bi\u1ebfn ng\u1eabu nhi\u00ean b\u1eb1ng qPCR th\u00ec c\u00f3 th\u1ec3 thay \u0111\u1ed5i n\u1ed3ng \u0111\u1ed9 t\u1eebng lo\u1ea1i dNTP cho ph\u00f9 h\u1ee3p v\u00e0 k\u1ebft h\u1ee3p Taq<\/em> polymerase kh\u00f4ng c\u00f3 ho\u1ea1t t\u00ednh s\u1eeda sai.<\/p>\n\n\n\n

3.5. Dung d\u1ecbch \u0111\u1ec7m v\u00e0 n\u1ed3ng \u0111\u1ed9 Mg2+<\/sup><\/strong><\/h3>\n\n\n\n

Dung d\u1ecbch \u0111\u1ec7m t\u1ea1o ra m\u1ed9t m\u00f4i tr\u01b0\u1eddng ph\u00f9 h\u1ee3p cho ph\u1ea3n \u1ee9ng qPCR di\u1ec5n ra, \u0111\u1eb7c bi\u1ec7t l\u00e0 ho\u1ea1t t\u00ednh DNA polymerase \u0111\u01b0\u1ee3c t\u1ed1i \u01b0u. \u0110\u1ec7m th\u01b0\u1eddng \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong ph\u1ea3n \u1ee9ng qPCR l\u00e0 Tris-KCl v\u1edbi pH dao \u0111\u1ed9ng t\u1eeb 8-9 gi\u00fap h\u00f2a tan v\u00e0 \u1ed5n \u0111\u1ecbnh DNA, ion K+<\/sup> trong \u0111\u1ec7m Tris h\u1ed7 tr\u1ee3 cho ho\u1ea1t \u0111\u1ed9ng g\u1eafn m\u1ed3i. Ngo\u00e0i ra, mu\u1ed1i ammonium sulfate (NH4<\/sub>)SO4<\/sub> c\u0169ng c\u00f3 th\u1ec3 \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng thay th\u1ebf KCl trong dung d\u1ecbch \u0111\u1ec7m, trong \u0111\u00f3 ion NH4<\/sub>+<\/sup> c\u00f3 kh\u1ea3 n\u0103ng l\u00e0m m\u1ea5t \u1ed5n \u0111\u1ecbnh li\u00ean k\u1ebft hydro gi\u1eefa c\u00e1c c\u1eb7p base b\u1eaft c\u1eb7p sai, gi\u00fap h\u1ed7 tr\u1ee3 t\u0103ng c\u01b0\u1eddng t\u00ednh \u0111\u1eb7c hi\u1ec7u cho ph\u1ea3n \u1ee9ng qPCR.<\/p>\n\n\n\n

MgCl2<\/sub> l\u00e0 mu\u1ed1i \u0111\u01b0\u1ee3c th\u00eam ri\u00eang l\u1ebb ho\u1eb7c ph\u1ed1i tr\u1ed9n v\u1edbi \u0111\u1ec7m Tris nh\u1eb1m b\u1ed5 sung ion Mg2+<\/sup> cho ho\u1ea1t \u0111\u1ed9ng c\u1ee7a DNA polymerase. Nguy\u00ean nh\u00e2n do DNA polymerase l\u00e0 m\u1ed9t holoenzyme n\u00ean c\u1ea7n m\u1ed9t cofactor g\u1eafn v\u00e0o trung t\u00e2m ho\u1ea1t \u0111\u1ed9ng th\u00ec m\u1edbi c\u00f3 ho\u1ea1t t\u00ednh v\u00e0 Mg2+<\/sup> \u0111\u00f3ng vai tr\u00f2 l\u00e0 cofactor c\u1ee7a enzyme n\u00e0y. Do \u0111\u00f3, n\u1ebfu qu\u00e1 \u00edt Mg2+<\/sup> th\u00ec DNA polymerase s\u1ebd kh\u00f4ng ho\u1ea1t \u0111\u1ed9ng ho\u1eb7c t\u1ed5ng h\u1ee3p k\u00e9m hi\u1ec7u qu\u1ea3, c\u00f2n n\u1ebfu qu\u00e1 nhi\u1ec1u Mg2+<\/sup> th\u00ec ph\u1ea3n \u1ee9ng PCR s\u1ebd kh\u00f4ng \u0111\u1ea3m b\u1ea3o \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u. N\u1ed3ng \u0111\u1ed9 MgCl2<\/sub> s\u1eed d\u1ee5ng s\u1ebd ph\u1ee5 thu\u1ed9c nhi\u1ec1u v\u00e0o DNA polymerase, dung d\u1ecbch \u0111\u1ec7m, dao \u0111\u1ed9ng t\u1eeb kho\u1ea3ng 0,5 \u0111\u1ebfn 3,5mM.<\/p>\n\n\n\n

3.6. Ch\u1ea5t ph\u00e1t hu\u1ef3nh quang s\u1eed d\u1ee5ng trong Real-time PCR<\/strong><\/h3>\n\n\n\n

C\u01a1 ch\u1ebf ph\u00e1t hu\u1ef3nh quang l\u00e0 \u0111i\u1ec3m \u0111\u1eb7c tr\u01b0ng t\u1ea1o n\u00ean s\u1ef1 kh\u00e1c bi\u1ec7t c\u1ee7a qPCR v\u00e0 PCR. Nh\u1edd v\u00e0o hi\u1ec7n t\u01b0\u1ee3ng ph\u00e1t x\u1ea1 hu\u1ef3nh quang c\u1ee7a m\u1ed9t s\u1ed1 ph\u00e2n t\u1eed ho\u00e1 h\u1ecdc \u1edf d\u1ea1ng c\u00f3 s\u1eb5n hay \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf \u0111\u1ec3 b\u1eaft v\u00e0o m\u1ee5c ti\u00eau gen c\u1ea7n ph\u00e1t hi\u1ec7n, h\u1ec7 th\u1ed1ng chuy\u00ean d\u1ee5ng s\u1ebd thu nh\u1eadn v\u00e0 ph\u00e2n t\u00edch t\u00edn hi\u1ec7u \u0111\u1ec3 tr\u1ea3 ra k\u1ebft qu\u1ea3 \u0111\u1ecbnh l\u01b0\u1ee3ng gen m\u1ee5c ti\u00eau. Hi\u1ec7n nay c\u00f3 hai c\u01a1 ch\u1ebf ph\u00e1t hu\u1ef3nh quang ch\u00ednh \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u00f3 l\u00e0 ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n v\u00e0 m\u1eabu d\u00f2.<\/p>\n\n\n\n

Ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n (Fluorescent Dye-based real-time PCR)<\/em><\/strong><\/p>\n\n\n\n

Ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n l\u00e0 nh\u1eefng ch\u1ea5t c\u00f3 \u00e1i l\u1ef1c cao v\u1edbi DNA m\u1ea1ch \u0111\u00f4i. Khi m\u1edbi b\u1eaft \u0111\u1ea7u ph\u1ea3n \u1ee9ng nh\u1eefng ch\u1ea5t n\u00e0y ph\u00e2n t\u00e1n trong dung d\u1ecbch v\u00e0 ph\u00e1t ra t\u00edn hi\u1ec7u \u1edf m\u1ee9c th\u1ea5p. Sau \u0111\u00f3, b\u1ea3n sao DNA b\u1eaft \u0111\u1ea7u \u0111\u01b0\u1ee3c t\u1ea1o ra th\u00ec ch\u1ea5t n\u00e0y s\u1ebd t\u1eadp trung l\u1ea1i v\u00e0 g\u1eafn ch\u00e8n v\u00e0o s\u1ee3i m\u1ea1ch \u0111\u00f4i, d\u1eabn \u0111\u1ebfn t\u00edn hi\u1ec7u ph\u00e1t hu\u1ef3nh quang s\u1ebd \u0111\u01b0\u1ee3c t\u0103ng l\u00ean (H\u00ecnh 1). <\/p>\n\n\n\n

Nh\u01b0 v\u1eady c\u00e0ng nhi\u1ec1u s\u1ee3i \u0111\u00f4i DNA \u0111\u01b0\u1ee3c t\u1ea1o ra th\u00ec c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang s\u1ebd t\u0103ng l\u00ean theo. C\u00e1c ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n ph\u1ed5 bi\u1ebfn hi\u1ec7n nay nh\u01b0 SYBR Green, EVA Green, EtBr,… v\u00e0 th\u01b0\u1eddng \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong c\u00e1c \u1ee9ng d\u1ee5ng ph\u00e1t hi\u1ec7n c\u00e1c \u0111\u1ed9t bi\u1ebfn hay x\u00e1c \u0111\u1ecbnh genotype.<\/p>\n\n\n\n

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H\u00ecnh 1. Nguy\u00ean l\u00fd ho\u1ea1t \u0111\u1ed9ng ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n trong real-time PCR (https:\/\/app.biorender.com\/<\/a>)<\/em><\/figcaption><\/figure>\n\n\n\n

<\/p>\n\n\n\n

Bi\u1ec3u \u0111\u1ed3 nhi\u1ec7t \u0111\u1ed9 n\u00f3ng ch\u1ea3y c\u1ee7a s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i<\/em><\/strong><\/p>\n\n\n\n

M\u1ed9t l\u01b0u \u00fd quan tr\u1ecdng khi s\u1eed d\u1ee5ng ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n \u0111\u00f3 l\u00e0 sau b\u01b0\u1edbc khu\u1ebfch \u0111\u1ea1i th\u00ec c\u1ea7n th\u1ef1c hi\u1ec7n th\u00eam b\u01b0\u1edbc ph\u00e2n t\u00edch nhi\u1ec7t \u0111\u1ed9 n\u00f3ng ch\u1ea3y. Nguy\u00ean nh\u00e2n l\u00e0 do ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n s\u1ebd g\u1eafn v\u00e0o b\u1ea5t k\u1ef3 ph\u00e2n t\u1eed DNA m\u1ea1ch \u0111\u00f4i n\u00e0o c\u00f3 m\u1eb7t trong dung d\u1ecbch m\u00e0 kh\u00f4ng c\u00f3 c\u01a1 ch\u1ebf g\u1eafn \u0111\u1eb7c hi\u1ec7u v\u1edbi tr\u00ecnh t\u1ef1 DNA m\u1ee5c ti\u00eau. Th\u1ebf n\u00ean, n\u1ebfu nh\u01b0 m\u1ed3i t\u1ea1o \u201cprimer dimer\u201d (hi\u1ec7n t\u01b0\u1ee3ng m\u1ed3i t\u1ef1 b\u1eaft c\u1eb7p v\u1edbi nhau thay v\u00ec g\u1eafn v\u1edbi m\u1ea1ch khu\u00f4n) th\u00ec k\u1ebft qu\u1ea3 s\u1ebd d\u1ec5 d\u1eabn \u0111\u1ebfn k\u1ebft lu\u1eadn l\u00e0 m\u1eabu d\u01b0\u01a1ng t\u00ednh gi\u1ea3 (kho\u1ea3ng 35-40 chu k\u1ef3)<\/p>\n\n\n\n

Nhi\u1ec7t \u0111\u1ed9 n\u00f3ng ch\u1ea3y c\u1ee7a s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i \u0111\u01b0\u1ee3c \u0111\u1ecbnh ngh\u0129a l\u00e0 t\u1ea1i m\u1ed9t \u0111i\u1ec3m nhi\u1ec7t \u0111\u1ed9 nh\u1ea5t \u0111\u1ecbnh c\u00f3 50% s\u1ee3i \u0111\u00f4i DNA b\u1ecb bi\u1ebfn t\u00ednh ho\u00e0n to\u00e0n. Gi\u00e1 tr\u1ecb Tm c\u1ee7a s\u1ea3n ph\u1ea9m gi\u00fap x\u00e1c \u0111\u1ecbnh xem t\u00edn hi\u1ec7u hu\u1ef3nh quang thu nh\u1eadn \u0111\u01b0\u1ee3c t\u1eeb ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i c\u00f3 ph\u1ea3i l\u00e0 c\u1ee7a s\u1ea3n ph\u1ea9m m\u1ee5c ti\u00eau hay do \u201cprimer dimer\u201d. Tm s\u1ebd ph\u1ee5 thu\u1ed9c v\u00e0o n\u1ed3ng \u0111\u1ed9 GC v\u00e0 \u0111\u1ed9 d\u00e0i tr\u00ecnh t\u1ef1, do v\u1eady Tm c\u1ee7a \u201cprimer dimer\u201d th\u01b0\u1eddng r\u1ea5t th\u1ea5p v\u00e0 r\u1ea5t d\u1ec5 ph\u00e2n bi\u1ec7t v\u1edbi Tm c\u1ee7a s\u1ea3n ph\u1ea9m PCR \u0111\u1eb7c hi\u1ec7u. Do v\u1eady, thi\u1ebft l\u1eadp ph\u1ea3n \u1ee9ng x\u00e1c \u0111\u1ecbnh Tm l\u00e0 c\u1ea7n thi\u1ebft khi s\u1eed d\u1ee5ng ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n.<\/p>\n\n\n\n

Ph\u1ea3n \u1ee9ng nhi\u1ec7t \u0111\u1ed9 n\u00f3ng ch\u1ea3y c\u1ee7a s\u1ea3n ph\u1ea9m Real-time PCR<\/em><\/strong><\/p>\n\n\n\n

Sau ph\u1ea3n \u1ee9ng real-time, s\u1ea3n ph\u1ea9m PCR s\u1ebd ti\u1ebfp t\u1ee5c tr\u1ea3i qua m\u1ed9t ch\u01b0\u01a1ng tr\u00ecnh x\u00e1c \u0111\u1ecbnh Tm. Ch\u01b0\u01a1ng tr\u00ecnh ph\u00e2n t\u00edch Tm b\u1eaft \u0111\u1ea7u v\u1edbi vi\u1ec7c n\u00e2ng nhi\u1ec7t l\u00ean 950<\/sup>C trong kho\u1ea3ng 15 gi\u00e2y \u0111\u1ec3 bi\u1ebfn t\u00ednh \u0111\u1ed3ng lo\u1ea1t c\u00e1c s\u1ee3i \u0111\u00f4i DNA, sau \u0111\u00f3 h\u1ea1 nhi\u1ec7t xu\u1ed1ng kho\u1ea3ng 55- 600<\/sup>C trong 15 gi\u00e2y \u0111\u1ec3 c\u00e1c s\u1ee3i n\u00e0y g\u1eafn b\u1ed5 sung l\u1ea1i. \u0110\u01b0\u1eddng cong n\u00f3ng ch\u1ea3y c\u1ee7a s\u1ea3n ph\u1ea9m PCR s\u1ebd b\u1eaft \u0111\u1ea7u \u0111\u01b0\u1ee3c quan s\u00e1t khi n\u00e2ng nhi\u1ec7t t\u1eeb 550<\/sup>C \u0111\u1ebfn 950<\/sup>C, \u1edf m\u1ed7i m\u1ee9c t\u0103ng 0,1-0,50<\/sup>C\/10 gi\u00e2y, t\u00edn hi\u1ec7u hu\u1ef3nh quang s\u1ebd ng\u00e0y c\u00e0ng gi\u1ea3m v\u00e0 m\u00e1y s\u1ebd ghi nh\u1eadn l\u1ea1i. <\/p>\n\n\n\n

M\u1ee9c t\u0103ng nhi\u1ec7t s\u1ebd t\u00f9y thu\u1ed9c v\u00e0o lo\u1ea1i ch\u1ea5t hu\u1ef3nh quang c\u0169ng nh\u01b0 d\u00f2ng m\u00e1y qPCR s\u1eed d\u1ee5ng. Sau khi ho\u00e0n th\u00e0nh ch\u01b0\u01a1ng tr\u00ecnh nhi\u1ec7t, gi\u00e1 tr\u1ecb Tm ghi nh\u1eadn \u0111\u01b0\u1ee3c s\u1ebd c\u00f3 d\u1ea1ng d\u1ed1c nghi\u00eang, do \u1edf t\u1eebng b\u01b0\u1edbc gia nhi\u1ec7t, DNA s\u1ebd bi\u1ebfn t\u00ednh d\u1eabn \u0111\u1ebfn c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang gi\u1ea3m d\u1ea7n. Tuy nhi\u00ean, Tm s\u1ebd \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh d\u1ec5 d\u00e0ng h\u01a1n th\u00f4ng qua vi\u1ec7c chuy\u1ec3n \u0111\u1ed5i bi\u1ec3u \u0111\u1ed3 sang d\u1ea1ng \u201cpeak\u201d (H\u00ecnh 2). <\/p>\n\n\n\n

Bi\u1ec3u \u0111\u1ed3 d\u1ea1ng n\u00e0y th\u1ec3 hi\u1ec7n gi\u00e1 tr\u1ecb m\u1ee9c \u0111\u1ed9 gi\u1ea3m hu\u1ef3nh quang (delta RFU) \u1ee9ng v\u1edbi m\u1ee9c t\u0103ng nhi\u1ec7t \u0111\u1ed9 (delta T), do v\u1eady gia nhi\u1ec7t \u0111\u1ebfn Tm th\u00ec t\u1ef7 l\u1ec7 delta RFU\/delta T s\u1ebd gia t\u0103ng \u0111\u1ed9t bi\u1ebfn t\u1ea1o m\u1ed9t peak r\u00f5 r\u1ec7t. Gi\u00e1 tr\u1ecb peak s\u1ebd t\u01b0\u01a1ng \u1ee9ng Tm \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh tr\u00ean tr\u1ee5c ho\u00e0nh c\u1ee7a s\u1ea3n ph\u1ea9m sau khu\u1ebfch \u0111\u1ea1i.<\/p>\n\n\n\n

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H\u00ecnh 2. \u0110\u1ed3 th\u1ecb bi\u1ec3u di\u1ec5n \u0111\u01b0\u1eddng cong n\u00f3ng ch\u1ea3y c\u1ee7a s\u1ea3n ph\u1ea9m PCR (Renter\u00eda-Sol\u00eds et al., 2020)<\/em><\/figcaption><\/figure>\n\n\n\n

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Ph\u00e2n t\u00edch HRM (high-resolution melt curve)<\/em><\/strong><\/p>\n\n\n\n

HRM l\u00e0 k\u1ef9 thu\u1eadt ph\u00e2n t\u00edch Tm c\u00f3 \u0111\u1ed9 ph\u00e2n gi\u1ea3i cao, gi\u00fap ph\u00e1t hi\u1ec7n v\u00e0 ph\u00e2n bi\u1ec7t c\u00e1c s\u1ea3n ph\u1ea9m PCR c\u00f3 \u0111\u1ed9 ch\u00eanh l\u1ec7ch Tm th\u1ea5p, n\u00f3i c\u00e1ch kh\u00e1c ph\u00e2n t\u00edch HRM c\u00f3 kh\u1ea3 n\u0103ng x\u00e1c \u0111\u1ecbnh c\u00e1c bi\u1ebfn th\u1ec3 trong tr\u00ecnh t\u1ef1 gen (\u0111\u1ed9t bi\u1ebfn \u0111i\u1ec3m hay SNPs). <\/p>\n\n\n\n

Ph\u00e2n t\u00edch n\u00e0y di\u1ec5n ra sau ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i v\u00e0 ph\u1ea7n m\u1ec1m m\u00e1y c\u0169ng s\u1ebd t\u1ef1 \u0111\u1ed9ng x\u1eed l\u00fd d\u00e0nh ri\u00eang cho HRM. V\u00ec HRM \u0111\u01b0\u1ee3c theo d\u00f5i \u201creal-time\u201d, do v\u1eady c\u0169ng s\u1ebd \u0111\u01b0a ra bi\u1ec3u \u0111\u1ed3 h\u00ecnh \u1ea3nh th\u1ef1c v\u1ec1 c\u00e1c \u0111\u1eb7c \u0111i\u1ec3m c\u1ee7a DNA \u0111ang \u0111\u01b0\u1ee3c th\u1eed nghi\u1ec7m khi ph\u1ea3n \u1ee9ng x\u1ea3y ra. Ph\u00e2n t\u00edch HRM l\u00e0 m\u1ed9t gi\u1ea3i ph\u00e1p thay th\u1ebf cho s\u00e0ng l\u1ecdc tr\u00ecnh t\u1ef1 b\u1eb1ng bi\u1ebfn t\u00ednh s\u1eafc k\u00fd l\u1ecfng hi\u1ec7u n\u0103ng cao dHPLC c\u1ee7a c\u00e1c bi\u1ebfn th\u1ec3 gen m\u1edbi. M\u1ed9t s\u1ed1 thu\u1ed1c nhu\u1ed9m th\u01b0\u01a1ng m\u1ea1i \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 th\u1ef1c hi\u1ec7n HRM l\u00e0 SYTO, Chromofy, LC Green, EvaGreen, \u2026 <\/p>\n\n\n\n

So v\u1edbi c\u00e1c c\u00f4ng ngh\u1ec7 ph\u00e1t hi\u1ec7n \u0111\u1ed9t bi\u1ebfn kh\u00e1c nh\u01b0 gi\u1ea3i tr\u00ecnh t\u1ef1, dHPLC v\u00e0 \u0111i\u1ec7n di gel gradient bi\u1ebfn t\u00ednh (Denaturing Gradient Gel Electrophoresis \u2013 DGGE), HRM cung c\u1ea5p m\u1ed9t ph\u01b0\u01a1ng ph\u00e1p d\u1ec5 d\u00e0ng h\u01a1n, \u00edt t\u1ed1n th\u1eddi gian h\u01a1n v\u00e0 ti\u1ebft ki\u1ec7m chi ph\u00ed. B\u00ean c\u1ea1nh \u0111\u00f3, ph\u00e2n t\u00edch HRM l\u00e0m gi\u1ea3m \u0111\u00e1ng k\u1ec3 kh\u1ea3 n\u0103ng nhi\u1ec5m ch\u00e9o t\u1eeb ph\u1ea3n \u1ee9ng PCR tr\u01b0\u1edbc. S\u1ef1 n\u00f3ng ch\u1ea3y c\u1ee7a s\u1ea3n ph\u1ea9m PCR HRM \u0111ang \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh ki\u1ec3u gen c\u00e1c bi\u1ebfn th\u1ec3 l\u01b0\u1ee1ng b\u1ed9i, ph\u00e2n t\u00edch methyl h\u00f3a DNA, x\u00e1c \u0111\u1ecbnh lo\u00e0i, \u0111\u1ecbnh lo\u1ea1i kh\u00e1ng nguy\u00ean b\u1ea1ch c\u1ea7u ng\u01b0\u1eddi hay ph\u00e1t hi\u1ec7n \u0111\u1ed9t bi\u1ebfn.<\/p>\n\n\n\n

M\u1eabu d\u00f2 (Fluorescent Probe-based real-time PCR)<\/em><\/strong><\/p>\n\n\n\n

M\u1eabu d\u00f2 (probe) l\u00e0 nh\u1eefng \u0111o\u1ea1n oligonucleotide s\u1ee3i \u0111\u01a1n c\u00f3 th\u1ec3 b\u1eaft c\u1eb7p \u0111\u1eb7c hi\u1ec7u v\u1edbi m\u1ed9t \u0111o\u1ea1n tr\u00ecnh t\u1ef1 tr\u00ean DNA m\u1ee5c ti\u00eau, bao g\u1ed3m \u0111\u1ea7u 5\u2019 g\u1eafn ch\u1ea5t ph\u00e1t hu\u1ef3nh quang (reporter) v\u00e0 \u0111\u1ea7u 3\u2019 g\u1eafn ch\u1ea5t h\u1ea5p ph\u1ee5 hu\u1ef3nh quang (quencher). Khi c\u1ea5u tr\u00fac c\u1ee7a m\u1eabu d\u00f2 c\u00f2n nguy\u00ean v\u1eb9n th\u00ec \u0111\u1ea7u quencher s\u1ebd l\u00e0m t\u1eaft t\u00edn hi\u1ec7u hu\u1ef3nh quang ph\u00e1t ra t\u1eeb \u0111\u1ea7u reporter n\u1ebfu kho\u1ea3ng c\u00e1ch hai \u0111\u1ea7u n\u00e0y \u0111\u1ee7 g\u1ea7n b\u1edfi c\u01a1 ch\u1ebf FRET, do d\u00f3 chi\u1ec1u d\u00e0i m\u1eabu d\u00f2 ch\u1ec9 n\u00ean t\u1eeb 18-22 nucleotide v\u00e0 quan tr\u1ecdng l\u00e0 reporter v\u00e0 quencher ph\u1ea3i t\u01b0\u01a1ng t\u00edch nhau v\u1ec1 b\u01b0\u1edbc s\u00f3ng k\u00edch th\u00edch v\u00e0 b\u01b0\u1edbc s\u00f3ng ph\u00e1t hu\u1ef3nh quang. <\/p>\n\n\n\n

V\u00ed d\u1ee5 n\u1ebfu reporter l\u00e0 FAM, TET, VIC th\u00ec quencher ph\u00f9 h\u1ee3p l\u00e0 TAMRA, Cy3, Cy5 v\u1edbi BHQ2. Khi ph\u1ea3n \u1ee9ng nh\u00e2n b\u1ea3n b\u1eaft \u0111\u1ea7u x\u1ea3y ra, c\u1ee5 th\u1ec3 l\u00e0 m\u1eabu d\u00f2 s\u1ebd g\u1eafn l\u00ean s\u1ee3i khu\u00f4n tr\u01b0\u1edbc m\u1ed3i v\u00e0 \u1edf v\u1ecb tr\u00ed sau m\u1ed3i theo chi\u1ec1u 5\u2019\u00e0 3\u2019, sau \u0111\u00f3 DNA polymerase kh\u1edfi \u0111\u1ed9ng qu\u00e1 tr\u00ecnh t\u1ed5ng h\u1ee3p s\u1ee3i m\u1edbi, khi g\u1eb7p m\u1eabu d\u00f2 s\u1ebd ho\u1ea1t h\u00f3a ho\u1ea1t t\u00ednh exonuclease \u0111\u1ec3 c\u1eaft c\u1ea5u tr\u00fac m\u1eabu d\u00f2. L\u00fac n\u00e0y, \u0111\u1ea7u reporter c\u1ee7a m\u1eabu d\u00f2 s\u1ebd t\u00e1ch r\u1eddi ho\u00e0n to\u00e0n v\u1edbi \u0111\u1ea7u quencher v\u00e0 ph\u00e1t hu\u1ef3nh quang do kh\u00f4ng c\u00f2n s\u1ef1 h\u1ea5p ph\u1ee5 \u00e1nh s\u00e1ng t\u1eeb quencher. <\/p>\n\n\n\n

H\u1ec7 th\u1ed1ng m\u00e1y real-time PCR s\u1ebd ghi nh\u1eadn t\u00edn hi\u1ec7u t\u1eeb reporter v\u00e0 hi\u1ec3n th\u1ecb tr\u00ean m\u00e0n h\u00ecnh m\u00e1y t\u00ednh. L\u01b0\u1ee3ng DNA \u0111\u01b0\u1ee3c khu\u1ebfch \u0111\u1ea1i c\u00e0ng nhi\u1ec1u, t\u00edn hi\u1ec7u hu\u1ef3nh quang thu \u0111\u01b0\u1ee3c s\u1ebd c\u00e0ng m\u1ea1nh.<\/p>\n\n\n\n

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H\u00ecnh 3. Nguy\u00ean l\u00fd ho\u1ea1t \u0111\u1ed9ng ch\u1ea5t hu\u1ef3nh quang m\u1eabu d\u00f2 trong real-time PCR (https:\/\/app.biorender.com\/)<\/em><\/figcaption><\/figure>\n\n\n\n

<\/p>\n\n\n\n

Taqman<\/a><\/strong> l\u00e0 m\u1eabu d\u00f2 ph\u1ed5 bi\u1ebfn \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong qPCR, m\u1eabu d\u00f2 n\u00e0y ho\u1ea1t \u0111\u1ed9ng d\u1ef1a tr\u00ean kh\u1ea3 n\u0103ng c\u1eaft exonuclease c\u1ee7a Taq polymerase. Ngo\u00e0i ra, c\u00f2n c\u00f3 TaqMan MGB (minor groove binder) v\u1edbi c\u1ea5u tr\u00fac \u0111\u1ea7u 3\u2019 ch\u1ee9a m\u1ed9t ph\u00e2n t\u1eed c\u00f3 kh\u1ea3 n\u0103ng b\u00e1m v\u00e0o r\u00e3nh xo\u1eafn nh\u1ecf c\u1ee7a DNA. Khi m\u1eabu d\u00f2 n\u00e0y b\u00e1m v\u00e0o tr\u00ecnh t\u1ef1 \u0111\u00edch, m\u1ed9t r\u00e3nh nh\u1ecf ng\u1eafn \u0111\u01b0\u1ee3c h\u00ecnh th\u00e0nh tr\u00ean DNA l\u00e0m t\u0103ng Tm, d\u1eabn \u0111\u1ebfn m\u1eabu d\u00f2 b\u00e1m ch\u1eb7t h\u01a1n hay n\u00f3i c\u00e1ch kh\u00e1c m\u1eabu d\u00f2 v\u1eabn b\u00e1m ch\u1eafc \u1edf nhi\u1ec7t \u0111\u1ed9 Ta<\/sub> c\u1ea7n thi\u1ebft cho m\u1ed3i m\u00e0 kh\u00f4ng c\u1ea7n t\u0103ng chi\u1ec1u d\u00e0i (H\u00ecnh 4).<\/p>\n\n\n\n

Ngo\u00e0i ra, c\u00f2n c\u00f3 m\u1ed9t s\u1ed1 lo\u1ea1i m\u1eabu d\u00f2 nh\u01b0 Molecular beacon, Scorpion primer. Theo \u0111\u00f3, Molecular beacon l\u00e0 m\u1ed9t DNA m\u1ea1ch \u0111\u01a1n d\u00e0i 20 \u2013 25 nu c\u0169ng \u0111\u01b0\u1ee3c \u0111\u00e1nh d\u1ea5u k\u00e9p \u1edf hai \u0111\u1ea7u nh\u01b0 Taqman, \u1edf tr\u1ea1ng th\u00e1i b\u00ecnh th\u01b0\u1eddng m\u1eabu d\u00f2 lo\u1ea1i n\u00e0y t\u1ea1o th\u00e0nh c\u1ea5u tr\u00fac k\u1eb9p t\u00f3c (H\u00ecnh 5). Vi\u1ec7c thi\u1ebft k\u1ebf molecular beacon y\u00eau c\u1ea7u 4 \u2013 6 nu \u1edf hai \u0111\u1ea7u c\u1ee7a n\u00f3 ph\u1ea3i t\u1ea1o th\u00e0nh c\u1ea5u tr\u00fac k\u1eb9p t\u00f3c, trong khi ph\u1ea7n loop khi bi\u1ebfn t\u00ednh th\u00e0nh d\u1ea1ng s\u1ee3i th\u1eb3ng s\u1ebd g\u1eafn b\u1ed5 sung v\u1edbi DNA m\u1ee5c ti\u00eau. <\/p>\n\n\n\n

\u1ede b\u01b0\u1edbc g\u1eafn m\u1ed3i c\u1ee7a ph\u1ea3n \u1ee9ng PCR, ph\u1ea7n loop c\u1ee7a Molecular beacon b\u00e1m v\u00e0o tr\u00ecnh t\u1ef1 \u0111\u00edch c\u1ee7a n\u00f3, l\u00e0m cho ph\u1ea7n th\u00e2n (stem) bi\u1ebfn t\u00ednh. L\u00fac n\u00e0y, reporter v\u00e0 quencher t\u00e1ch xa nhau ra l\u00e0m cho m\u00e1y ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c t\u00edn hi\u1ec7u hu\u1ef3nh quang ph\u00e1t ra t\u1eeb reporter. S\u1ed1 l\u01b0\u1ee3ng tr\u00ecnh t\u1ef1 \u0111\u00edch qua t\u1eebng chu k\u1ef3 ph\u1ea3n \u1ee9ng \u0111\u01b0\u1ee3c th\u1ec3 hi\u1ec7n qua c\u01b0\u1eddng \u0111\u1ed9 ph\u00e1t quang. <\/p>\n\n\n\n

Trong khi \u0111\u00f3, Scorpion primer l\u00e0 c\u00e1c ph\u00e2n t\u1eed hai ch\u1ee9c n\u0103ng, c\u00f3 m\u1ed3i \u0111\u01b0\u1ee3c g\u1eafn c\u1ed9ng h\u00f3a tr\u1ecb v\u1edbi m\u1eabu d\u00f2. Ph\u00e2n t\u1eed n\u00e0y c\u0169ng ch\u1ee9a m\u1ed9t ch\u1ea5t ph\u00e1t quang reporter v\u00e0 m\u1ed9t quencher d\u1eadp t\u1eaft t\u00edn hi\u1ec7u (H\u00ecnh 6). Nguy\u00ean l\u00fd \u0111\u1ecbnh l\u01b0\u1ee3ng s\u1eed d\u1ee5ng Scorpion primer l\u00e0 gi\u1ed1ng v\u1edbi Taqman v\u00e0 molecular beacon, tuy nhi\u00ean c\u1ea5u tr\u00fac m\u1eabu d\u00f2 n\u00e0y ph\u1ee9c t\u1ea1p v\u00e0 kh\u00f3 thi\u1ebft k\u1ebf h\u01a1n.<\/p>\n\n\n\n

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H\u00ecnh 4. Ch\u1ea5t hu\u1ef3nh quang TaqMan MGB (https:\/\/www.elitechgroup.com\/)<\/em><\/figcaption><\/figure>\n\n\n\n
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H\u00ecnh 5. Ch\u1ea5t hu\u1ef3nh quang Molecular Beacon (Artika et al., 2022)<\/em><\/figcaption><\/figure>\n\n\n\n
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H\u00ecnh 6. Ch\u1ea5t hu\u1ef3nh quang Scorpion primer (Artika et al., 2022)<\/em><\/figcaption><\/figure>\n\n\n\n

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3.7. Real-time PCR mix (qPCR mix)<\/strong><\/h3>\n\n\n\n

Tr\u01b0\u1edbc \u0111\u00e2y, ph\u1ea3n \u1ee9ng qPCR \u0111\u01b0\u1ee3c ph\u1ed1i tr\u1ed9n l\u1ea1i t\u1eeb c\u00e1c th\u00e0nh ph\u1ea7n ri\u00eang l\u1ebb bao g\u1ed3m Taq DNA polymerase, dung d\u1ecbch \u0111\u1ec7m, MgCl2<\/sub>, dNTP v\u00e0 n\u01b0\u1edbc sinh h\u1ecdc ph\u00e2n t\u1eed \u0111\u01b0\u1ee3c cung c\u1ea5p trong c\u00f9ng m\u1ed9t b\u1ed9 s\u1ea3n ph\u1ea9m, sau \u0111\u00f3 m\u1edbi b\u1ed5 sung c\u00e1c th\u00e0nh ph\u1ea7n c\u00f2n thi\u1ebfu nh\u01b0 m\u1ed3i v\u00e0 ch\u1ea5t hu\u1ef3nh quang. C\u00e1c th\u00e0nh ph\u1ea7n s\u1ebd \u0111\u01b0\u1ee3c pha theo n\u1ed3ng \u0111\u1ed9 \u0111\u01b0\u1ee3c khuy\u1ebfn c\u00e1o b\u1edfi nh\u00e0 s\u1ea3n xu\u1ea5t nh\u01b0ng v\u1eabn c\u00f3 th\u1ec3 linh ho\u1ea1t thay \u0111\u1ed5i d\u1ef1a tr\u00ean n\u1ed3ng \u0111\u1ed9 \u0111\u1ec1 ngh\u1ecb \u0111\u1ec3 \u0111\u1ea1t hi\u1ec7u qu\u1ea3 cho ph\u1ea3n \u1ee9ng. <\/p>\n\n\n\n

B\u00ean c\u1ea1nh \u0111\u00f3, vi\u1ec7c ph\u1ed1i tr\u1ed9n \u0111\u01a1n l\u1ebb c\u00f2n c\u00f3 \u01b0u \u0111i\u1ec3m l\u00e0 Taq polymerase s\u1ebd \u0111\u01b0\u1ee3c b\u1ea3o qu\u1ea3n t\u1ed1t trong dung d\u1ecbch glycerol c\u00f3 \u0111\u1ed9 b\u1ec1n cao h\u01a1n do kh\u00f4ng b\u1ecb \u0111\u00f4ng v\u00e0 r\u00e3 \u0111\u00f4ng nhi\u1ec1u l\u1ea7n v\u00e0 c\u00f3 th\u1ec3 c\u1eaft gi\u1ea3m l\u01b0\u1ee3ng enzyme s\u1eed d\u1ee5ng. Tuy nhi\u00ean, c\u0169ng gi\u1ed1ng nh\u01b0 PCR, ph\u1ea3n \u1ee9ng real-time PCR \u0111\u01b0\u1ee3c thi\u1ebft l\u1eadp t\u1eeb c\u00e1c th\u00e0nh ph\u1ea7n \u0111\u01a1n l\u1ebb s\u1ebd c\u1ea7n nhi\u1ec1u th\u1eddi gian v\u00e0 t\u0103ng sai s\u1ed1 do thao t\u00e1c. Do v\u1eady, hi\u1ec7n nay mix cho ph\u1ea3n \u1ee9ng qPCR c\u0169ng \u0111\u00e3 \u0111\u01b0\u1ee3c ph\u00e1t tri\u1ec3n \u0111\u1ec3 \u0111\u00e1p \u1ee9ng \u0111a d\u1ea1ng nhu c\u1ea7u s\u1eed d\u1ee5ng.<\/p>\n\n\n\n

Real-time PCR mix l\u00e0 h\u1ed7n h\u1ee3p ch\u1ee9a c\u00e1c th\u00e0nh ph\u1ea7n c\u1ea7n thi\u1ebft cho ph\u1ea3n \u1ee9ng real-time PCR v\u1edbi n\u1ed3ng \u0111\u1ed9 \u0111\u1eadm \u0111\u1eb7c (th\u01b0\u1eddng l\u00e0 2X hay 5X). T\u01b0\u01a1ng t\u1ef1 PCR, th\u00e0nh ph\u1ea7n real-time PCR mix v\u1ec1 c\u01a1 b\u1ea3n bao g\u1ed3m: polymerase, dNTP, Mg2+<\/sup> v\u00e0 \u0111\u1ec7m t\u1ed1i \u01b0u ph\u1ea3n \u1ee9ng. Khi s\u1eed d\u1ee5ng th\u00ec ngo\u00e0i vi\u1ec7c cho khu\u00f4n DNA v\u00e0 m\u1ed3i nh\u01b0 PCR th\u00ec ch\u1ea5t ph\u00e1t hu\u1ef3nh quang (ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n ho\u1eb7c probe) s\u1ebd \u0111\u01b0\u1ee3c th\u00eam v\u00e0o mix \u0111\u1ec3 th\u1ef1c hi\u1ec7n ph\u1ea3n \u1ee9ng. <\/p>\n\n\n\n

Tr\u01b0\u1edbc \u0111\u00e2y, real-time PCR mix chia l\u00e0m hai lo\u1ea1i ch\u00ednh: b\u1ed5 sung SYBR Green v\u00e0 b\u1ed5 sung probe. SYBR Green l\u00e0 ch\u1ea5t ph\u00e1t hu\u1ef3nh quang g\u1eafn ch\u00e8n \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng ph\u1ed5 bi\u1ebfn trong qPCR, ch\u1ea5t n\u00e0y \u0111\u01b0\u1ee3c cho v\u00e0o trong mix s\u1eb5n, ng\u01b0\u1eddi d\u00f9ng ch\u1ec9 c\u1ea7n b\u1ed5 sung c\u1eb7p m\u1ed3i v\u00e0 m\u1eabu l\u00e0 c\u00f3 th\u1ec3 ti\u1ebfn h\u00e0nh qPCR. \u0110\u1ed1i v\u1edbi probe, ng\u01b0\u1eddi d\u00f9ng ph\u1ea3i thi\u1ebft k\u1ebf probe sao cho tr\u00ecnh t\u1ef1 probe b\u1eaft c\u1eb7p v\u1edbi gen \u0111\u00edch tr\u01b0\u1edbc m\u1ed3i v\u00e0 nhi\u1ec7t \u0111\u1ed9 b\u1eaft c\u1eb7p ph\u1ea3i cao h\u01a1n m\u1ed3i t\u1eeb 8-100<\/sup>C. <\/p>\n\n\n\n

C\u00e1c th\u00e0nh ph\u1ea7n trong mix \u0111\u01b0\u1ee3c thay \u0111\u1ed5i linh ho\u1ea1t \u0111\u1ec3 ph\u00f9 h\u1ee3p v\u1edbi t\u1eebng lo\u1ea1i real-time PCR c\u0169ng nh\u01b0 t\u00e1c nh\u00e2n m\u1ee5c ti\u00eau. S\u1ef1 c\u1ea3i ti\u1ebfn v\u00e0 t\u1ed1i \u01b0u h\u00f3a real-time PCR mix s\u1ebd t\u01b0\u01a1ng \u1ee9ng v\u1edbi s\u1ef1 ph\u00e1t tri\u1ec3n c\u1ee7a k\u1ef9 thu\u1eadt n\u00e0y v\u00e0 \u0111\u00e1p \u1ee9ng nhu c\u1ea7u s\u1eed d\u1ee5ng ng\u00e0y c\u00e0ng gia t\u0103ng. C\u1ee5 th\u1ec3 nh\u01b0 c\u00e1c b\u1ed9 kit \u0111\u1ec3 ch\u1ea9n \u0111o\u00e1n m\u1ed9t s\u1ed1 t\u00e1c nh\u00e2n g\u00e2y c\u00e1c b\u1ec7nh truy\u1ec1n nhi\u1ec5m, c\u00e1c b\u1ed9 kit real-time PCR cung c\u1ea5p c\u00e1c mix c\u00f3 s\u1eb5n m\u1ed3i-probe \u0111\u00e3 \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf \u0111\u1eb7c hi\u1ec7u cho v\u00f9ng gen c\u1ee7a t\u00e1c nh\u00e2n nh\u1eb1m r\u00fat ng\u1eafn th\u1eddi gian x\u00e9t nghi\u1ec7m m\u00e0 v\u1eabn mang l\u1ea1i \u0111\u1ed9 ch\u00ednh x\u00e1c cao.<\/p>\n\n\n\n

Ngo\u00e0i k\u1ef9 thu\u1eadt qPCR truy\u1ec1n th\u1ed1ng th\u00ec hi\u1ec7n nay c\u00e1c kit \u0111\u00e3 c\u1ea3i ti\u1ebfn \u0111\u1ec3 \u1ee9ng d\u1ee5ng \u0111\u01b0\u1ee3c tr\u00ean nhi\u1ec1u k\u1ef9 thu\u1eadt nh\u01b0 multiplex qPCR hay RT-qPCR. T\u01b0\u01a1ng t\u1ef1 nh\u01b0 multiplex PCR mix, multiple qPCR mix ch\u1ec9 kh\u00e1c vi\u1ec7c b\u1ed5 sung probe hay ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n cho vi\u1ec7c \u0111\u1ecdc t\u00edn hi\u1ec7u hu\u1ef3nh quang. C\u00e1c mix c\u1ee7a k\u1ef9 thu\u1eadt n\u00e0y th\u01b0\u1eddng mang nhi\u1ec1u \u0111\u1eb7c t\u00ednh nh\u01b0 s\u1eed d\u1ee5ng cho \u0111a t\u00e1c nh\u00e2n (2-4 t\u00e1c nh\u00e2n), c\u00e1c ch\u1ea5t hu\u1ef3nh quang \u0111\u01b0\u1ee3c t\u1ed1i \u01b0u \u0111\u1ec3 gi\u1ea3m thi\u1ec3u s\u1ef1 nhi\u1ec5u t\u00edn hi\u1ec7u thu \u0111\u01b0\u1ee3c gi\u1eefa c\u00e1c k\u00eanh m\u00e0u (Cross-talk) c\u0169ng nh\u01b0 \u0111\u1ed9 nh\u1ea1y t\u01b0\u01a1ng \u0111\u01b0\u01a1ng qPCR singleplex. <\/p>\n\n\n\n

Vi\u1ec7c th\u1ef1c hi\u1ec7n multiplex qPCR k\u1ebft h\u1ee3p mix pha s\u1eb5n s\u1ebd gi\u00fap ti\u1ebft ki\u1ec7m th\u1eddi gian khi thi\u1ebft l\u1eadp ph\u1ea3n \u1ee9ng v\u00e0 d\u1ec5 d\u00e0ng \u0111\u1ed1i chi\u1ebfu ph\u00e2n t\u00edch k\u1ebft qu\u1ea3. Ngo\u00e0i c\u00e1c multiplex qPCR mix s\u1eed d\u1ee5ng chung cho c\u00e1c lo\u1ea1i t\u00e1c nh\u00e2n n\u00f3i chung th\u00ec hi\u1ec7n nay m\u1ed9t s\u1ed1 s\u1ea3n ph\u1ea9m mix \u0111\u01b0\u1ee3c s\u1ea3n xu\u1ea5t ri\u00eang cho vi\u1ec7c ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng c\u00e1c t\u00e1c nh\u00e2n c\u1ee5 th\u1ec3. V\u1edbi nh\u1eefng lo\u1ea1i mix n\u00e0y, trong th\u00e0nh ph\u1ea7n \u0111\u00e3 b\u1ed5 sung c\u00e1c c\u1eb7p m\u1ed3i v\u00e0 probe \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf \u0111\u1ea3m b\u1ea3o s\u1ef1 b\u1eaft c\u1eb7p \u0111\u1eb7c hi\u1ec7u v\u1edbi v\u00f9ng gen nh\u1ea5t \u0111\u1ecbnh c\u1ee7a t\u00e1c nh\u00e2n, nh\u01b0 v\u1eady khi s\u1eed d\u1ee5ng ng\u01b0\u1eddi d\u00f9ng ch\u1ec9 c\u1ea7n b\u1ed5 sung khu\u00f4n m\u1eabu (DNA\/RNA).<\/p>\n\n\n\n

\u0110\u1ed1i v\u1edbi k\u1ef9 thu\u1eadt Quantitative reverse transcripton (RT-qPCR), hi\u1ec7n nay c\u00f3 hai mix ph\u1ed5 bi\u1ebfn \u0111\u00f3 l\u00e0 One-step v\u00e0 two-step. Tr\u01b0\u1edbc \u0111\u00e2y, k\u1ef9 thu\u1eadt RT-qPCR s\u1ebd tr\u1ea3i qua hai b\u01b0\u1edbc ri\u00eang bi\u1ec7t \u0111\u00f3 l\u00e0 t\u1ed5ng h\u1ee3p cDNA v\u00e0 real-time PCR c\u00f9ng v\u1edbi vi\u1ec7c s\u1eed d\u1ee5ng hai mix t\u01b0\u01a1ng \u1ee9ng k\u00e8m theo (two-step). \u01afu \u0111i\u1ec3m \u0111\u00f3 l\u00e0 th\u00e0nh ph\u1ea7n m\u1ed7i lo\u1ea1i mix s\u1ebd t\u1ed1i \u01b0u cho ph\u1ea3n \u1ee9ng v\u00e0 c\u00f3 th\u1ec3 tr\u1eef RNA sau khi k\u1ebft th\u00fac ph\u1ea3n \u1ee9ng. Tuy nhi\u00ean, vi\u1ec7c th\u1ef1c hi\u1ec7n l\u1ea7n l\u01b0\u1ee3t t\u1eebng ph\u1ea3n \u1ee9ng s\u1ebd m\u1ea5t th\u1eddi gian v\u00e0 nhi\u1ec1u thao t\u00e1c th\u1ef1c hi\u1ec7n, nguy c\u01a1 nhi\u1ec5m cao v\u00e0 nh\u1ea5t l\u00e0 kh\u00f3 \u1ee9ng d\u1ee5ng trong ch\u1ea9n \u0111o\u00e1n b\u1ec7nh c\u1ea7n x\u00e1c \u0111\u1ecbnh t\u00e1c nh\u00e2n nhanh ch\u00f3ng. <\/p>\n\n\n\n

Trong khi \u0111\u00f3, mix one-step RT-qPCR cho ph\u00e9p tr\u1ef1c ti\u1ebfp th\u1ef1c hi\u1ec7n k\u1ebft h\u1ee3p hai ph\u1ea3n \u1ee9ng t\u1ed5ng h\u1ee3p cDNA v\u00e0 real-time PCR trong c\u00f9ng \u1ed1ng ph\u1ea3n \u1ee9ng, gi\u00fap ti\u1ebft ki\u1ec7m th\u1eddi gian v\u00e0 h\u1ea1n ch\u1ebf thao t\u00e1c g\u00e2y nhi\u1ec5m, tuy nhi\u00ean khi g\u1ed9p ph\u1ea3n \u1ee9ng th\u00ec c\u00e1c th\u00e0nh ph\u1ea7n s\u1ebd kh\u00f4ng \u0111\u01b0\u1ee3c ho\u1ea1t \u0111\u1ed9ng t\u1ed1i \u01b0u cho t\u1eebng lo\u1ea1i ph\u1ea3n \u1ee9ng, v\u00ed d\u1ee5 nh\u01b0 th\u00e0nh ph\u1ea7n \u0111\u1ec7m ph\u1ea3i d\u00f9ng chung cho Taq polymerase v\u00e0 reverse transcriptase hay kh\u00f4ng th\u1ec3 l\u01b0u tr\u1eef RNA l\u1ea1i sau khi ph\u1ea3n \u1ee9ng k\u1ebft th\u00fac. Do v\u1eady t\u00f9y thu\u1ed9c v\u00e0o m\u1ee5c \u0111\u00edch s\u1eed d\u1ee5ng m\u00e0 l\u1ef1a ch\u1ecdn mix RT-qPCR ph\u00f9 h\u1ee3p.<\/p>\n\n\n\n

B\u00ean c\u1ea1nh t\u1ed1i \u01b0u h\u00f3a c\u00e1c th\u00e0nh ph\u1ea7n ph\u1ea3n \u1ee9ng, nh\u00e0 s\u1ea3n xu\u1ea5t mix qPCR c\u00f2n b\u1ed5 sung m\u1ed9t s\u1ed1 ch\u1ea5t t\u0103ng c\u01b0\u1eddng (enhancer) v\u00e0 ch\u1ea5t \u1ed5n \u0111\u1ecbnh ph\u1ea3n \u1ee9ng (stabilizer). Trong khi, ch\u1ea5t t\u0103ng c\u01b0\u1eddng h\u1ed7 tr\u1ee3 cho vi\u1ec7c nh\u00e2n b\u1ea3n nh\u1eefng v\u00f9ng gen kh\u00f3 (gi\u00e0u GC) ho\u1eb7c khi m\u1eabu DNA c\u00f3 ch\u1ee9a ch\u1ea5t \u1ee9c ch\u1ebf th\u00ec ch\u1ea5t \u1ed5n \u0111\u1ecbnh \u0111\u01b0\u1ee3c b\u1ed5 sung nh\u1eb1m b\u1ea3o v\u1ec7 ho\u1ea1t \u0111\u1ed9ng c\u1ee7a Taq polymerase sau t\u00e1i r\u00e3 \u0111\u00f4ng nhi\u1ec1u l\u1ea7n.<\/p>\n\n\n\n

Ngo\u00e0i ra, t\u00f9y d\u00f2ng m\u00e1y m\u00e0 trong th\u00e0nh ph\u1ea7n mix s\u1ebd c\u00f3 th\u00eam ch\u1ea5t hu\u1ef3nh quang b\u1ecb \u0111\u1ed9ng (passive reference dye) l\u00e0 ROX (6-corboxy-X-rhodmine). Kh\u00e1c v\u1edbi SYBR Green hay probe, t\u00edn hi\u1ec7u hu\u1ef3nh quang t\u1eeb ROX g\u1ecdi l\u00e0 \u201cb\u1ecb \u0111\u1ed9ng\u201d \u0111\u00f3 l\u00e0 do t\u00edn hi\u1ec7u t\u1eeb ch\u1ea5t n\u00e0y kh\u00f4ng ph\u1ea3n \u00e1nh s\u1ef1 khu\u1ebfch \u0111\u1ea1i DNA m\u1ee5c ti\u00eau m\u00e0 t\u00edn hi\u1ec7u thu nh\u1eadn \u0111\u01b0\u1ee3c l\u00e0 t\u1eeb b\u1ea5t k\u1ef3 th\u1ee9 g\u00ec kh\u00e1c c\u00f3 th\u1ec3 l\u00e0m thay \u0111\u1ed5i t\u00edn hi\u1ec7u hu\u1ef3nh quang t\u1ed5ng (m\u1ed3i, probe, tu\u00fdp, \u2026). T\u00edn hi\u1ec7u t\u1eeb ROX s\u1ebd \u0111\u01b0\u1ee3c thu nh\u1eadn b\u1eb1ng m\u1ed9t k\u00eanh m\u00e0u ri\u00eang v\u00e0 song song \u0111\u1ed9c l\u1eadp v\u1edbi t\u00edn hi\u1ec7u c\u1ee7a s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i. <\/p>\n\n\n\n

Vi\u1ec7c d\u00f9ng ROX s\u1ebd gi\u00fap chu\u1ea9n h\u00f3a t\u00edn hi\u1ec7u hu\u1ef3nh quang, tr\u00e1nh c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang n\u1ec1n \u1ea3nh h\u01b0\u1edfng sai l\u1ec7ch \u0111\u1ebfn t\u00edn hi\u1ec7u hu\u1ef3nh quang th\u1eadt s\u1ef1 c\u1ee7a s\u1ea3n ph\u1ea9m khu\u1ebfch \u0111\u1ea1i \u0111\u1ed3ng th\u1eddi \u0111\u1ea3m b\u1ea3o kh\u1ea3 n\u0103ng l\u1eb7p l\u1ea1i th\u00ed nghi\u1ec7m gi\u1eefa c\u00e1c tu\u00fdp m\u1eabu. Vi\u1ec7c l\u1ef1a ch\u1ecdn mix c\u00f3 ROX c\u1ea7n \u0111\u01b0\u1ee3c ch\u00fa \u00fd ph\u1ea3i ph\u00f9 h\u1ee3p v\u1edbi lo\u1ea1i m\u00e1y qPCR \u0111ang s\u1eed d\u1ee5ng \u0111\u1ec3 tr\u00e1nh l\u00e0m \u1ea3nh h\u01b0\u1edfng \u0111\u1ebfn k\u1ebft qu\u1ea3 \u0111o thu nh\u1eadn \u0111\u01b0\u1ee3c.<\/p>\n\n\n\n

H\u1ea7u h\u1ebft c\u00e1c mix hi\u1ec7n nay \u0111\u1ec1u \u1edf d\u1ea1ng l\u1ecfng, ti\u1ec7n l\u1ee3i cho vi\u1ec7c s\u1eed d\u1ee5ng. Tuy nhi\u00ean, nh\u00e0 s\u1ea3n xu\u1ea5t g\u1eb7p m\u1ed9t s\u1ed1 kh\u00f3 kh\u0103n trong vi\u1ec7c b\u1ea3o qu\u1ea3n v\u00e0 v\u1eadn chuy\u1ec3n c\u00e1c \u1ed1ng mix l\u1ecfng, d\u1ec5 g\u1eb7p t\u00ecnh tr\u1ea1ng bung n\u1eafp hay r\u00f2 r\u1ec9 dung d\u1ecbch do r\u01a1i v\u1ee1. B\u00ean c\u1ea1nh \u0111\u00f3, mix l\u1ecfng y\u00eau c\u1ea7u tr\u1eef trong thi\u1ebft b\u1ecb chuy\u00ean d\u1ee5ng \u1edf nhi\u1ec7t \u0111\u1ed9 t\u1eeb -200<\/sup>C \u0111\u1ebfn -700<\/sup>C (n\u1ebfu c\u1ea7n tr\u1eef trong th\u1eddi gian d\u00e0i) \u0111\u1ec3 duy tr\u00ec ho\u1ea1t t\u00ednh, do v\u1eady vi\u1ec7c n\u00e0y m\u1eb7c nhi\u00ean tr\u1edf th\u00e0nh \u00e1p l\u1ef1c v\u1ec1 chi ph\u00ed b\u1ea3o qu\u1ea3n kh\u00f4ng ch\u1ec9 trong s\u1eed d\u1ee5ng m\u00e0 c\u1ea3 v\u1eadn chuy\u1ec3n cho nh\u00e0 s\u1ea3n xu\u1ea5t v\u00e0 ng\u01b0\u1eddi s\u1eed d\u1ee5ng. <\/p>\n\n\n\n

Do v\u1eady, c\u00f4ng ngh\u1ec7 \u0111\u00f4ng kh\u00f4 hay g\u1ea7n \u0111\u00e2y l\u00e0 s\u1ea5y kh\u00f4 \u0111ang b\u01b0\u1edbc \u0111\u1ea7u \u0111\u01b0\u1ee3c \u1ee9ng d\u1ee5ng v\u00e0o k\u1ef9 thu\u1eadt qPCR. \u0110\u00f4ng kh\u00f4 l\u00e0 m\u1ed9t qu\u00e1 tr\u00ecnh kh\u1eed n\u01b0\u1edbc \u1edf nhi\u1ec7t \u0111\u1ed9 th\u1ea5p ch\u1ee7 y\u1ebfu \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 \u1ed5n \u0111\u1ecbnh c\u00e1c d\u01b0\u1ee3c ch\u1ea5t sinh h\u1ecdc kh\u00f4ng b\u1ec1n v\u1edbi nhi\u1ec7t c\u00f3 trong dung d\u1ecbch n\u01b0\u1edbc [Hansen et al., 2015]. <\/p>\n\n\n\n

Tuy nhi\u00ean, m\u00e1y \u0111\u00f4ng kh\u00f4 hi\u1ec7n nay v\u1eabn l\u00e0 thi\u1ebft b\u1ecb \u0111\u1eaft ti\u1ec1n n\u00ean kh\u00f3 kh\u0103n v\u1ec1 chi ph\u00ed cho c\u00e1c ph\u00f2ng th\u00ed nghi\u1ec7m. Ngo\u00e0i ra, qu\u00e1 tr\u00ecnh \u0111\u00f4ng kh\u00f4 s\u1eed d\u1ee5ng nhi\u1ec7t \u0111\u1ed9 cao \u0111\u1ec3 bay h\u01a1i ho\u00e0n to\u00e0n h\u01a1i n\u01b0\u1edbc c\u00f3 th\u1ec3 g\u00e2y h\u1ecfng mix, \u0111\u1eb7c bi\u1ec7t l\u00e0 ho\u1ea1t t\u00ednh c\u1ee7a poplymerase. V\u1edbi c\u00f4ng ngh\u1ec7 s\u1ea5y kh\u00f4, m\u1ed9t t\u1ee7 s\u1ea5y b\u00ecnh th\u01b0\u1eddng \u1edf c\u00e1c ph\u00f2ng th\u00ed nghi\u1ec7m c\u00f3 th\u1ec3 \u0111\u00e1p \u1ee9ng l\u00e0m kh\u00f4 mix \u1edf kho\u1ea3ng 700<\/sup>C. Vi\u1ec7c s\u1eed d\u1ee5ng mix kh\u00f4 c\u0169ng \u0111\u00e3 \u0111\u01b0\u1ee3c nghi\u00ean c\u1ee9u ch\u1ee9ng minh t\u00ednh \u1ed5n \u0111\u1ecbnh v\u00e0 hi\u1ec7u qu\u1ea3 t\u01b0\u01a1ng \u0111\u01b0\u01a1ng mix l\u1ecfng.<\/p>\n\n\n\n

Nh\u1eefng c\u1ea3i ti\u1ebfn v\u1ec1 mix qPCR hi\u1ec7n nay \u0111\u1ec1u h\u01b0\u1edbng \u0111\u1ebfn vi\u1ec7c thi\u1ebft l\u1eadp ph\u1ea3n \u1ee9ng hi\u1ec7u qu\u1ea3, nhanh ch\u00f3ng, ti\u1ebft ki\u1ec7m v\u1ec1 m\u1eb7t chi ph\u00ed v\u00e0 th\u1eddi gian. \u0110\u1ed1i v\u1edbi nh\u1eefng ph\u00f2ng th\u00ed nghi\u1ec7m tr\u01b0\u1eddng h\u1ecdc hay trung t\u00e2m, th\u00f4ng th\u01b0\u1eddng s\u1ebd s\u1eed d\u1ee5ng lo\u1ea1i th\u00e0nh ph\u1ea7n \u0111\u01a1n l\u1ebb \u0111\u1ec3 t\u1ed1i \u01b0u h\u00f3a quy tr\u00ecnh nghi\u00ean c\u1ee9u, c\u00f2n ri\u00eang \u0111\u1ed1i v\u1edbi c\u00e1c ph\u00f2ng th\u00ed nghi\u1ec7m d\u1ecbch v\u1ee5 hay b\u1ec7nh vi\u1ec7n, th\u1eddi gian v\u00e0 \u0111\u1ed9 t\u00e1i l\u1eb7p c\u1ee7a quy tr\u00ecnh l\u00e0 c\u1ef1c k\u1ef3 quan tr\u1ecdng. Ch\u00ednh v\u00ec v\u1eady, \u0111\u1ea1i \u0111a s\u1ed1 c\u00e1c b\u1ec7nh vi\u1ec7n, trung t\u00e2m d\u1ecbch v\u1ee5 s\u1ebd \u01b0u ti\u00ean d\u00f9ng qPCR mix thay v\u00ec c\u00e1c th\u00e0nh ph\u1ea7n ph\u1ea3n \u1ee9ng \u0111\u01a1n l\u1ebb nh\u01b0 tr\u01b0\u1edbc \u0111\u00e2y.<\/p>\n\n\n\n

4. M\u1ed9t s\u1ed1 thu\u1eadt ng\u1eef trong ph\u00e2n t\u00edch k\u1ebft qu\u1ea3 real-time PCR<\/strong><\/h2>\n\n\n\n

\u0110\u1ed1i v\u1edbi k\u1ef9 thu\u1eadt qPCR, do t\u00ednh ch\u1ea5t \u201creal-time\u201d n\u00ean ng\u01b0\u1eddi l\u00e0m c\u00f3 th\u1ec3 theo d\u00f5i qu\u00e1 tr\u00ecnh ph\u1ea3n \u1ee9ng di\u1ec5n ra \u0111\u01b0\u1ee3c th\u1ec3 hi\u1ec7n tr\u00ean h\u1ec7 th\u1ed1ng m\u00e1y t\u00ednh. T\u00edn hi\u1ec7u hu\u1ef3nh quang thu nh\u1eadn \u0111\u01b0\u1ee3c s\u1ebd chuy\u1ec3n \u0111\u1ed5i th\u00e0nh d\u1ea1ng bi\u1ec3u \u0111\u1ed3 \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n khu\u1ebfch \u0111\u1ea1i, bi\u1ec3u \u0111\u1ed3 n\u00e0y s\u1ebd bao g\u1ed3m c\u00e1c th\u00f4ng s\u1ed1 v\u1ec1 t\u00edn hi\u1ec7u n\u1ec1n (background), \u0111\u01b0\u1eddng n\u1ec1n (baseline) v\u00e0 chu k\u1ef3 ng\u01b0\u1ee1ng (cycle threshold- Ct) (H\u00ecnh 7). Trong \u0111\u00f3:<\/p>\n\n\n\n

T\u00edn hi\u1ec7u n\u1ec1n (Background)<\/strong>: l\u00e0 c\u00e1c t\u00edn hi\u1ec7u hu\u1ef3nh quang n\u1ec1n \u0111\u01b0\u1ee3c t\u1ea1o th\u00e0nh kh\u00f4ng li\u00ean quan \u0111\u1ebfn ph\u1ea3n \u1ee9ng real-time PCR. T\u00edn hi\u1ec7u n\u00e0y th\u01b0\u1eddng do \u0111\u1ea7u reporter c\u1ee7a probe ph\u00e1t ra, v\u1ec1 l\u00fd thuy\u1ebft th\u00ec \u1edf \u0111i\u1ec1u ki\u1ec7n b\u00ecnh th\u01b0\u1eddng quencher s\u1ebd h\u1ea5p th\u1ee5 h\u1ebft t\u00edn hi\u1ec7u do reporter ph\u00e1t ra, nh\u01b0ng th\u1ef1c t\u1ebf quencher kh\u00f4ng th\u1ec3 h\u1ea5p th\u1ee5 100% t\u00edn hi\u1ec7u do reporter ph\u00e1t ra n\u00ean trong h\u1ea7u h\u1ebft c\u00e1c ph\u1ea3n \u1ee9ng real-time PCR \u0111\u1ec1u xu\u1ea5t hi\u1ec7n t\u00edn hi\u1ec7u n\u1ec1n. Ngo\u00e0i ra, m\u1ed7i lo\u1ea1i probe kh\u00e1c nhau, nh\u00e0 s\u1ea3n xu\u1ea5t kh\u00e1c nhau th\u00ec t\u00edn hi\u1ec7u n\u1ec1n c\u1ee7a t\u1eebng probe s\u1ebd kh\u00e1c nhau. <\/p>\n\n\n\n

Ng\u00e0y nay, c\u00e1c h\u00e3ng t\u1ed5ng h\u1ee3p probe c\u0169ng \u0111\u00e3 thi\u1ebft k\u1ebf ra nhi\u1ec1u c\u00e1ch \u0111\u1ec3 h\u1ea1n ch\u1ebf t\u00edn hi\u1ec7u n\u1ec1n n\u00e0y nh\u01b0 dual quencher,\u2026 V\u1ec1 tr\u01b0\u1eddng h\u1ee3p s\u1eed d\u1ee5ng SYBR Green, ban \u0111\u1ea7u khi ch\u01b0a c\u00f3 tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau th\u00ec ch\u1ea5t n\u00e0y v\u1eabn ph\u00e1t hu\u1ef3nh quang trong dung d\u1ecbch tuy nhi\u00ean do ph\u00e2n t\u00e1n n\u00ean c\u01b0\u1eddng \u0111\u1ed9 s\u1ebd y\u1ebfu v\u00e0 t\u1ea1o th\u00e0nh t\u00edn hi\u1ec7u n\u1ec1n cho ph\u1ea3n \u1ee9ng. Nh\u1eefng chu k\u1ef3 \u0111\u1ea7u c\u1ee7a real-time, n\u1ed3ng \u0111\u1ed9 ban \u0111\u1ea7u c\u1ee7a m\u1eabu th\u1ea5p n\u00ean c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang ph\u00e1t ra kh\u00f4ng \u0111\u1ee7 \u0111\u1ec3 m\u00e1y real-time PCR c\u00f3 th\u1ec3 ghi nh\u1eadn \u0111\u01b0\u1ee3c v\u00e0 giai \u0111o\u1ea1n n\u00e0y th\u01b0\u1eddng ch\u1ec9 ghi nh\u1eadn \u0111\u01b0\u1ee3c t\u00edn hi\u1ec7u n\u1ec1n c\u1ee7a m\u1eabu.<\/p>\n\n\n\n

\u0110\u01b0\u1eddng n\u1ec1n (<\/strong>Baseline<\/strong>)<\/strong>: l\u00e0 \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n m\u1ee9c \u0111\u1ed9 t\u00edn hi\u1ec7u trong su\u1ed1t nh\u1eefng chu k\u1ef3 \u0111\u1ea7u c\u1ee7a real-time PCR. Vi\u1ec7c thi\u1ebft l\u1eadp baseline l\u00e0 b\u01b0\u1edbc quan tr\u1ecdng trong ph\u00e2n t\u00edch k\u1ebft qu\u1ea3 real-time PCR do baseline kh\u00f4ng c\u1ed1 \u0111\u1ecbnh, gi\u00e1 tr\u1ecb baseline s\u1ebd \u1ea3nh h\u01b0\u1edfng nhi\u1ec1u \u0111\u1ebfn k\u1ebft qu\u1ea3 cu\u1ed1i c\u00f9ng. N\u1ebfu \u0111i\u1ec1u ch\u1ec9nh gi\u00e1 tr\u1ecb baseline qu\u00e1 th\u1ea5p, k\u1ebft qu\u1ea3 s\u1ebd r\u1ea5t d\u1ec5 thu nh\u1eadn t\u00edn hi\u1ec7u nhi\u1ec5u (d\u01b0\u01a1ng t\u00ednh gi\u1ea3), ng\u01b0\u1ee3c l\u1ea1i gi\u00e1 tr\u1ecb baseline qu\u00e1 cao, k\u1ebft qu\u1ea3 s\u1ebd c\u00f3 \u0111\u1ed9 ch\u00ednh x\u00e1c th\u1ea5p v\u00ec b\u1ecf qua c\u00e1c m\u1eabu d\u01b0\u01a1ng y\u1ebfu (\u00e2m t\u00ednh gi\u1ea3). V\u00ec v\u1eady, baseline n\u00ean \u0111\u01b0\u1ee3c \u0111i\u1ec1u ch\u1ec9nh kho\u1ea3ng 5% so v\u1edbi gi\u00e1 tr\u1ecb t\u00edn hi\u1ec7u hu\u1ef3nh quang cao nh\u1ea5t c\u1ee7a m\u1eabu<\/p>\n\n\n\n

Ng\u01b0\u1ee1ng (Threshold)<\/strong>: l\u00e0 m\u1ee9c t\u00edn hi\u1ec7u m\u00e0 \u1edf \u0111\u00f3 n\u00f3 ph\u1ea3n \u00e1nh s\u1ef1 gia t\u0103ng t\u00edn hi\u1ec7u c\u00f3 \u00fd ngh\u0129a v\u1ec1 m\u1eb7t th\u1ed1ng k\u00ea, gi\u00fap ph\u00e2n bi\u1ec7t t\u00edn hi\u1ec7u khu\u1ebfch \u0111\u1ea1i v\u01b0\u1ee3t qua kh\u1ecfi t\u00edn hi\u1ec7u n\u1ec1n. Ng\u01b0\u1eddi ta th\u01b0\u1eddng thi\u1ebft l\u1eadp gi\u00e1 tr\u1ecb ng\u01b0\u1ee1ng d\u1ef1a v\u00e0o \u0111\u01b0\u1eddng n\u1ec1n ho\u1eb7c m\u1ee9c t\u00edn hi\u1ec7u th\u1ef1c t\u1ebf c\u1ee7a m\u1eabu. Gi\u00e1 tr\u1ecb ng\u01b0\u1ee1ng \u0111\u01b0\u1ee3c thi\u1ebft l\u1eadp t\u01b0\u01a1ng t\u1ef1 nh\u01b0 baseline.<\/p>\n\n\n\n

Chu k\u1ef3 ng\u01b0\u1ee1ng (<\/strong>Ct<\/strong> –<\/strong> threshold cycle)<\/strong>: l\u00e0 s\u1ed1 chu k\u1ef3 m\u00e0 t\u1ea1i \u0111i\u1ec3m \u0111\u00f3 t\u00edn hi\u1ec7u hu\u1ef3nh quang c\u1ee7a ph\u1ea3n \u1ee9ng v\u01b0\u1ee3t l\u00ean c\u1eaft \u0111\u01b0\u1eddng ng\u01b0\u1ee1ng, thay \u0111\u1ed5i t\u1eeb \u00e2m t\u00ednh (kh\u00f4ng th\u1ec3 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c) sang d\u01b0\u01a1ng t\u00ednh (c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c). Gi\u00e1 tr\u1ecb chu k\u1ef3 ng\u01b0\u1ee1ng (Ct) ph\u1ea3n \u00e1nh l\u01b0\u1ee3ng gen quan t\u00e2m trong giai \u0111o\u1ea1n \u0111\u1ea7u c\u1ee7a ph\u1ea3n \u1ee9ng v\u00e0 t\u1ef7 l\u1ec7 ngh\u1ecbch v\u1edbi m\u1ee9c bi\u1ec3u hi\u1ec7n t\u01b0\u01a1ng \u0111\u1ed1i ban \u0111\u1ea7u c\u1ee7a gen quan t\u00e2m. N\u1ebfu gi\u00e1 tr\u1ecb Ct c\u00e0ng nh\u1ecf c\u00f3 ngh\u0129a l\u00e0 n\u1ed3ng \u0111\u1ed9 gen quan t\u00e2m ban \u0111\u1ea7u c\u00e0ng l\u1edbn v\u00e0 ng\u01b0\u1ee3c l\u1ea1i.<\/p>\n\n\n\n

Ch\u1ea5t hu\u1ef3nh quang b\u1ecb \u0111\u1ed9ng (Passive reference dye)<\/strong>: th\u01b0\u1eddng \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong real-time PCR \u0111\u1ec3 chu\u1ea9n h\u00f3a t\u00edn hi\u1ec7u hu\u1ef3nh quang c\u1ee7a reporter v\u00e0 hi\u1ec7u ch\u1ec9nh s\u1ef1 dao \u0111\u1ed9ng v\u1ec1 t\u00edn hi\u1ec7u hu\u1ef3nh quang kh\u00f4ng t\u1ea1o th\u00e0nh do ph\u1ea3n \u1ee9ng PCR gi\u1eefa c\u00e1c gi\u1ebfng. Kh\u00f4ng ph\u1ea3i m\u00e1y n\u00e0o c\u0169ng c\u00f3 ch\u1ebf \u0111\u1ed9 n\u00e0y do v\u1eady c\u1ea7n xem k\u1ef9 th\u00f4ng tin c\u1ee7a m\u00e1y \u0111\u1ec3 ch\u1ecdn lo\u1ea1i h\u00f3a ch\u1ea5t sao cho ph\u00f9 h\u1ee3p.<\/p>\n\n\n\n

Gi\u00e1 tr\u1ecb ng\u01b0\u1ee1ng k\u1ef9 thu\u1eadt (The cycle cut-off value)<\/strong>: gi\u00e1 tr\u1ecb n\u00e0y th\u01b0\u1eddng \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh \u0111\u1ed1i v\u1edbi x\u00e9t nghi\u1ec7m \u0111\u1ecbnh t\u00ednh, d\u1ef1a v\u00e0o gi\u1edbi h\u1ea1n ph\u00e1t hi\u1ec7n c\u1ee7a ph\u01b0\u01a1ng ph\u00e1p x\u00e9t nghi\u1ec7m (LOD). Tr\u01b0\u1edbc khi x\u00e9t nghi\u1ec7m, c\u1ea7n ph\u1ea3i x\u00e1c \u0111\u1ecbnh kho\u1ea3ng tuy\u1ebfn t\u00ednh c\u1ee7a ph\u01b0\u01a1ng ph\u00e1p d\u1ef1a tr\u00ean gi\u1edbi h\u1ea1n tr\u00ean v\u00e0 gi\u1edbi h\u1ea1n d\u01b0\u1edbi c\u1ee7a ph\u01b0\u01a1ng ph\u00e1p. Gi\u1edbi h\u1ea1n d\u01b0\u1edbi l\u00e0 n\u1ed3ng \u0111\u1ed9 t\u1ed1i thi\u1ec3u m\u00e0 ph\u01b0\u01a1ng ph\u00e1p c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c m\u1ed9t c\u00e1ch ch\u00ednh x\u00e1c, d\u1ef1a v\u00e0o \u0111\u00e2y ng\u01b0\u1eddi ta s\u1ebd thi\u1ebft l\u1eadp gi\u00e1 tr\u1ecb Ct cut-off. <\/p>\n\n\n\n

Th\u00f4ng th\u01b0\u1eddng c\u00e1c chu tr\u00ecnh nhi\u1ec7t c\u1ee7a m\u1ed9t ph\u1ea3n \u1ee9ng real-time PCR s\u1ebd \u0111\u01b0\u1ee3c c\u00e0i \u0111\u1eb7t t\u1eeb 40-45 chu k\u1ef3. Nh\u1eefng m\u1eabu xu\u1ea5t hi\u1ec7n Ct \u0111\u01b0\u1ee3c coi l\u00e0 d\u01b0\u01a1ng t\u00ednh, kh\u00f4ng xu\u1ea5t hi\u1ec7n Ct \u0111\u01b0\u1ee3c coi l\u00e0 \u00e2m t\u00ednh. C\u00e1c kit x\u00e9t nghi\u1ec7m tr\u00ean th\u1ecb tr\u01b0\u1eddng hi\u1ec7n nay th\u01b0\u1eddng c\u00f3 gi\u00e1 tr\u1ecb Ct cut-off trong kho\u1ea3ng 35-37. Nh\u1eefng m\u1eabu n\u00e0o c\u00f3 gi\u00e1 tr\u1ecb Ct nh\u1ecf h\u01a1n ho\u1eb7c b\u1eb1ng gi\u00e1 tr\u1ecb Ct cut-off \u0111\u01b0\u1ee3c coi l\u00e0 c\u00f3 \u0111\u1ed9 tin c\u1eady cao, c\u00f2n m\u1eabu n\u00e0o c\u00f3 gi\u00e1 tr\u1ecb Ct l\u1edbn h\u01a1n Ct cut-off \u0111\u01b0\u1ee3c coi l\u00e0 nghi ng\u1edd c\u1ea7n ch\u1ea1y l\u1ea1i \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh ho\u1eb7c k\u1ebft lu\u1eadn \u00e2m t\u00ednh.<\/p>\n\n\n\n

C\u00e1c m\u1eabu d\u01b0\u01a1ng t\u00ednh mu\u1ed9n c\u00f3 Ct t\u1eeb 38-45, \u0111\u01b0\u1ee3c \u0111\u00e1nh gi\u00e1 l\u00e0 d\u01b0\u01a1ng t\u00ednh gi\u1ea3 ho\u1eb7c n\u1ed3ng \u0111\u1ed9 qu\u00e1 th\u1ea5p b\u1ed9 kit kh\u00f4ng th\u1ec3 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c, nguy\u00ean nh\u00e2n c\u00f3 th\u1ec3 do s\u1ef1 b\u1eaft c\u1eb7p kh\u00f4ng \u0111\u1eb7c hi\u1ec7u, nhi\u1ec5m ch\u00e9o ho\u1eb7c s\u1ef1 tho\u00e1i h\u00f3a c\u1ee7a Taq polymerase, probe,\u2026<\/p>\n\n\n\n

Vi\u1ec7c \u0111\u1ecdc k\u1ebft qu\u1ea3 real time PCR s\u1ebd t\u00f9y v\u00e0o ph\u01b0\u01a1ng ph\u00e1p s\u1eed d\u1ee5ng (lo\u1ea1i m\u1eabu d\u00f2, lo\u1ea1i ch\u1ee9ng n\u1ed9i hay lo\u1ea1i m\u00e1y s\u1eed d\u1ee5ng).<\/p>\n\n\n\n

\"real-time
H\u00ecnh 7. \u0110\u01b0\u1eddng bi\u1ec3u di\u1ec5n c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang trong real-time PCR (https:\/\/geneticeducation.co.in\/)<\/em><\/figcaption><\/figure>\n\n\n\n

<\/p>\n\n\n\n

\u0110\u01b0\u1eddng chu\u1ea9n (Standard curve) <\/strong>Kh\u00e1c v\u1edbi PCR, ngo\u00e0i h\u1ec7 th\u1ed1ng m\u1eabu \u0111\u1ed1i ch\u1ee9ng, x\u00e9t nghi\u1ec7m qPCR c\u00f2n c\u00f3 m\u1ed9t th\u00f4ng s\u1ed1 \u0111\u1ec3 ki\u1ec3m so\u00e1t thao t\u00e1c v\u00e0 hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i, \u0111\u00f3 l\u00e0 \u0111\u01b0\u1eddng chu\u1ea9n. Theo \u0111\u1ecbnh ngh\u0129a, \u0111\u01b0\u1eddng chu\u1ea9n \u0111\u01b0\u1ee3c x\u00e2y d\u1ef1ng t\u1eeb gi\u00e1 tr\u1ecb chu k\u1ef3 ng\u01b0\u1ee1ng (Ct) c\u1ee7a m\u1ed9t d\u00e3y c\u00e1c \u1ed1ng ch\u1ee9a DNA c\u00f3 n\u1ed3ng \u0111\u1ed9 tuy\u1ebfn t\u00ednh \u0111\u00e3 \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh tr\u01b0\u1edbc (th\u01b0\u1eddng pha lo\u00e3ng theo b\u1eadc 10 nh\u01b0 101<\/sup>, 102<\/sup>, 103<\/sup>,\u2026). N\u1ed3ng \u0111\u1ed9 \u0111\u01b0\u1ee3c ch\u1ecdn cho vi\u1ec7c x\u00e2y d\u1ef1ng \u0111\u01b0\u1eddng chu\u1ea9n ph\u1ea3i bao qu\u00e1t \u0111\u01b0\u1ee3c n\u1ed3ng \u0111\u1ed9 d\u1ef1 ki\u1ebfn c\u1ee7a DNA m\u1ee5c ti\u00eau c\u00f3 trong m\u1eabu th\u1eed nghi\u1ec7m. \u0110\u01b0\u1eddng chu\u1ea9n bao g\u1ed3m tr\u1ee5c x (n\u1ed3ng \u0111\u1ed9 chu\u1ea9n) v\u00e0 tr\u1ee5c y (gi\u00e1 tr\u1ecb Ct). <\/p>\n\n\n\n

C\u00e1c th\u00f4ng s\u1ed1 c\u1ee7a \u0111\u01b0\u1eddng chu\u1ea9n nh\u01b0 h\u1ec7 s\u1ed1 tuy\u1ebfn t\u00ednh (R2<\/sup>), hi\u1ec7u su\u1ea5t khu\u1ebfch \u0111\u1ea1i (E%) v\u00e0 \u0111\u1ed9 d\u1ed1c (slope) \u0111\u01b0\u1ee3c m\u00e1y t\u00ednh to\u00e1n d\u1ef1a tr\u00ean k\u1ebft qu\u1ea3 ghi nh\u1eadn th\u1ef1c t\u1ebf, gi\u00fap x\u00e1c \u0111\u1ecbnh ch\u1ea5t l\u01b0\u1ee3ng c\u1ee7a m\u1ed9t ph\u1ea3n \u1ee9ng qPCR (H\u00ecnh 8), c\u1ee5 th\u1ec3:<\/p>\n\n\n\n

– H\u1ec7 s\u1ed1 tuy\u1ebfn t\u00ednh (R2<\/sup>): ph\u1ea3n \u00e1nh m\u1ee9c \u0111\u1ed9 tuy\u1ebfn t\u00ednh c\u1ee7a c\u00e1c gi\u00e1 tr\u1ecb trong \u0111\u01b0\u1eddng chu\u1ea9n, R2<\/sup> l\u00fd t\u01b0\u1edfng l\u00e0 ti\u1ebfn \u0111\u1ebfn 1, th\u1ef1c t\u1ebf gi\u00e1 tr\u1ecb t\u1ed1i \u0111a ch\u1ec9 t\u1edbi m\u1ee9c 0,999.<\/p>\n\n\n\n

– Hi\u1ec7u su\u1ea5t khu\u1ebfch \u0111\u1ea1i (E%): ph\u1ea3n \u00e1nh m\u1ee9c \u0111\u1ed9 hi\u1ec7u qu\u1ea3 c\u1ee7a ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i. E% l\u00fd t\u01b0\u1edfng l\u00e0 100%, \u0111\u00f3 l\u00e0 khi DNA m\u1ee5c ti\u00eau t\u0103ng g\u1ea5p \u0111\u00f4i sau m\u1ed7i chu k\u1ef3 nhi\u1ec7t trong giai \u0111o\u1ea1n l\u0169y th\u1eeba c\u1ee7a ph\u1ea3n \u1ee9ng. Tuy nhi\u00ean tr\u00ean th\u1ef1c t\u1ebf, hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i s\u1ebd c\u00f3 s\u1ef1 sai l\u1ec7ch so v\u1edbi gi\u00e1 tr\u1ecb l\u00fd t\u01b0\u1edfng, c\u1ee5 th\u1ec3 dao \u0111\u1ed9ng trong kho\u1ea3ng 90-110%. <\/p>\n\n\n\n

N\u1ebfu E th\u1ea5p, nguy\u00ean do c\u00f3 th\u1ec3 l\u00e0 \u0111\u1ed9 nh\u1ea1y c\u1ee7a m\u1ed3i ch\u01b0a cao hay ph\u1ea3n \u1ee9ng b\u1ecb \u1ee9c ch\u1ebf, n\u1ebfu E cao c\u00f3 th\u1ec3 l\u00e0 m\u1ed3i b\u1eaft c\u1eb7p \u0111\u1eb7c hi\u1ec7u. M\u1ed9t s\u1ed1 y\u1ebfu t\u1ed1 v\u1ec1 th\u00e0nh ph\u1ea7n nh\u01b0 chi\u1ec1u d\u00e0i gen \u0111\u00edch, th\u00e0nh ph\u1ea7n GC, c\u00e1c c\u1ea5u tr\u00fac th\u1ee9 c\u1ea5p h\u00ecnh th\u00e0nh hay thao t\u00e1c ng\u01b0\u1eddi th\u1ef1c hi\u1ec7n s\u1ebd \u1ea3nh h\u01b0\u1edfng \u0111\u1ebfn E%. <\/p>\n\n\n\n

Trong nghi\u00ean c\u1ee9u, thao t\u00e1c pha lo\u00e3ng m\u1eabu chu\u1ea9n c\u0169ng s\u1ebd \u1ea3nh h\u01b0\u1edfng \u0111\u1ebfn hi\u1ec7u su\u1ea5t. V\u00ed d\u1ee5, khi pha lo\u00e3ng m\u1eabu chu\u1ea9n t\u1eeb n\u1ed3ng \u0111\u1ed9 cao \u0111\u1ebfn th\u1ea5p s\u1ebd d\u1ec5 g\u1eb7p ph\u1ea3i tr\u01b0\u1eddng h\u1ee3p th\u1ec3 t\u00edch m\u1eabu n\u1ed3ng \u0111\u1ed9 cao tr\u01b0\u1edbc \u0111\u00f3 s\u1ebd c\u00f2n d\u00ednh \u1edf \u0111\u1ea7u tip d\u1eabn \u0111\u1ebfn l\u01b0\u1ee3ng DNA th\u1ef1c t\u1ebf \u1edf c\u00e1c \u1ed1ng pha lo\u00e3ng sau \u0111\u00f3 s\u1ebd c\u00f3 cao h\u01a1n d\u1ef1 ki\u1ebfn, do v\u1eady E c\u00f3 th\u1ec3 cao h\u01a1n 100%. Tr\u01b0\u1eddng h\u1ee3p ng\u01b0\u1ee3c l\u1ea1i n\u1ebfu khi dung d\u1ecbch kh\u00f4ng \u0111\u01b0\u1ee3c \u0111\u1ea9y ra h\u1ebft c\u0169ng l\u00e0m gi\u1ea3m gi\u00e1 tr\u1ecb E. <\/p>\n\n\n\n

– \u0110\u1ed9 d\u1ed1c (slope): \u0111\u01b0\u1ee3c coi l\u00e0 th\u01b0\u1edbc \u0111o hi\u1ec7u su\u1ea5t khu\u1ebfch \u0111\u1ea1i. N\u00f3i c\u00e1ch kh\u00e1c \u0111\u01b0\u1eddng chu\u1ea9n \u0111\u01b0\u1ee3c x\u00e2y d\u1ef1ng v\u1edbi c\u00e1c m\u1eabu chu\u1ea9n n\u1ebfu c\u00e1ch nhau qua \u0111\u1ed9 pha lo\u00e3ng h\u1ec7 s\u1ed1 10 th\u00ec hi\u1ec7u qu\u1ea3 khu\u1ebfch \u0111\u1ea1i E \u0111\u01b0\u1ee3c t\u00ednh theo c\u00f4ng th\u1ee9c 10-1\/slope<\/sup> v\u1edbi slope ch\u00ednh l\u00e0 \u0111\u1ed9 \u0111\u1ed1c c\u1ee7a \u0111\u01b0\u1eddng bi\u1ec3u di\u1ec5n chu\u1ea9n. V\u1edbi hi\u1ec7u qu\u1ea3 l\u00fd t\u01b0\u1edfng l\u00e0 s\u1ea3n ph\u1ea9m b\u1ea3n sao t\u0103ng l\u00ean g\u1ea5p \u0111\u00f4i so v\u1edbi khu\u00f4n ban \u0111\u1ea7u th\u00ec 10-1\/slope<\/sup> <\/sup>= 2, nh\u01b0 v\u1eady slope l\u00fd t\u01b0\u1edfng l\u00e0 -3,32 t\u01b0\u01a1ng \u0111\u01b0\u01a1ng E=100%. Tuy nhi\u00ean, th\u1ef1c t\u1ebf kh\u00f3 c\u00f3 th\u1ec3 \u0111\u1ea1t \u0111\u01b0\u1ee3c \u0111i\u1ec1u ki\u1ec7n nh\u01b0 v\u1eady, do \u0111\u00f3 slope th\u01b0\u1eddng dao \u0111\u1ed9ng  trong kho\u1ea3ng t\u1eeb -3,58 \u0111\u1ebfn -3,10.<\/p>\n\n\n\n

Sau ph\u1ea3n \u1ee9ng, d\u1ef1a v\u00e0o ph\u01b0\u01a1ng tr\u00ecnh y=ax+b c\u1ee7a \u0111\u01b0\u1eddng chu\u1ea9n s\u1ebd gi\u00fap x\u00e1c \u0111\u1ecbnh \u0111\u01b0\u1ee3c n\u1ed3ng \u0111\u1ed9 DNA m\u1ee5c ti\u00eau c\u00f3 trong m\u1eabu. Nh\u01b0 v\u1eady \u0111\u01b0\u1eddng chu\u1ea9n l\u00e0 m\u1ed9t th\u00f4ng s\u1ed1 \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh h\u00e0m l\u01b0\u1ee3ng b\u1ea3n sao DNA c\u00f3 trong m\u1eabu v\u00e0 \u0111\u00e1nh gi\u00e1 hi\u1ec7u qu\u1ea3 c\u1ee7a ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i. Hi\u1ec7n nay, tr\u00ean th\u1ecb tr\u01b0\u1eddng m\u1ed9t s\u1ed1 kit qPCR \u0111\u00e3 cung c\u1ea5p s\u1eb5n c\u00e1c \u1ed1ng n\u1ed3ng \u0111\u1ed9 chu\u1ea9n \u0111\u1ec3 \u0111\u1ea3m b\u1ea3o vi\u1ec7c x\u00e2y d\u1ef1ng \u0111\u01b0\u1eddng chu\u1ea9n ch\u00ednh x\u00e1c, kh\u00f4ng b\u1ecb \u1ea3nh h\u01b0\u1edfng nhi\u1ec1u b\u1edfi thao t\u00e1c ng\u01b0\u1eddi th\u1ef1c hi\u1ec7n.<\/p>\n\n\n\n

\"real-time
H\u00ecnh 8. \u0110\u1ed3 th\u1ecb ph\u00e2n t\u00edch c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang v\u00e0 \u0111\u01b0\u1eddng chu\u1ea9n (https:\/\/www.merckmillipore.com\/)<\/em><\/figcaption><\/figure>\n\n\n\n

<\/p>\n\n\n\n

5. C\u00e1c bi\u1ebfn th\u1ec3 c\u1ee7a k\u1ef9 thu\u1eadt real-time PCR<\/strong><\/h2>\n\n\n\n

K\u1ef9 thu\u1eadt real-time PCR hi\u1ec7n nay \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng ph\u1ed5 bi\u1ebfn \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh s\u1ef1 hi\u1ec7n di\u1ec7n hay \u0111o t\u1ea3i l\u01b0\u1ee3ng c\u1ee7a t\u00e1c nh\u00e2n m\u1ee5c ti\u00eau (vi r\u00fat, vi khu\u1ea9n,\u2026) th\u00f4ng qua ph\u00e1t hi\u1ec7n v\u1eadt li\u1ec7u di truy\u1ec1n c\u1ee7a ch\u00fang l\u00e0 DNA hay RNA. Trong b\u00e0i vi\u1ebft n\u00e0y s\u1ebd \u0111\u1ec1 c\u1eadp \u0111\u1ebfn c\u00e1c k\u1ef9 thu\u1eadt real-time bao g\u1ed3m qPCR \u0111\u1ecbnh t\u00ednh, qPCR \u0111\u1ecbnh l\u01b0\u1ee3ng, multiplex RT-qPCR v\u00e0 droplet digital PCR<\/p>\n\n\n\n

5.1. Real-time PCR \u0111\u1ecbnh t\u00ednh<\/strong><\/h3>\n\n\n\n

K\u1ef9 thu\u1eadt qPCR \u0111\u1ecbnh t\u00ednh t\u01b0\u01a1ng t\u1ef1 PCR, d\u00f9ng \u0111\u1ec3 x\u00e1c nh\u1eadn ch\u00ednh x\u00e1c v\u00e0 nhanh ch\u00f3ng s\u1ef1 c\u00f3 m\u1eb7t c\u1ee7a t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh \u0111\u01b0\u1ee3c nghi ng\u1edd c\u00f3 trong m\u1eabu b\u1ec7nh ph\u1ea9m, \u0111\u1eb7c bi\u1ec7t l\u00e0 b\u1ec7nh truy\u1ec1n nhi\u1ec5m, gi\u00fap h\u1ea1n ch\u1ebf s\u1ef1 l\u00e2y lan b\u1ec7nh trong c\u1ed9ng \u0111\u1ed3ng. Tuy nhi\u00ean, \u0111i\u1ec3m kh\u00e1c bi\u1ec7t l\u00e0 ph\u00e2n t\u00edch \u0111\u01b0\u1eddng c\u01b0\u1eddng \u0111\u1ed9 t\u00edn hi\u1ec7u hu\u1ef3nh quang ngay tr\u00ean m\u00e1y t\u00ednh m\u00e0 kh\u00f4ng c\u1ea7n th\u1ef1c hi\u1ec7n \u0111i\u1ec7n di nh\u01b0 PCR, \u0111i\u1ec1u n\u00e0y gi\u00fap vi\u1ec7c tr\u1ea3 k\u1ebft qu\u1ea3 nhanh ch\u00f3ng v\u00e0 k\u1ecbp th\u1eddi t\u00ecm ra nguy\u00ean nh\u00e2n g\u00e2y b\u1ec7nh. <\/p>\n\n\n\n

V\u1edbi qPCR \u0111\u1ecbnh t\u00ednh, th\u01b0\u1eddng s\u1eed d\u1ee5ng ch\u1ea5t hu\u1ef3nh quang g\u1eafn ch\u00e8n (th\u01b0\u1eddng l\u00e0 SYBR Green) hay probe (th\u01b0\u1eddng l\u00e0 Taqman) t\u00f9y v\u00e0o \u0111i\u1ec1u ki\u1ec7n kinh ph\u00ed c\u1ee7a ng\u01b0\u1eddi s\u1eed d\u1ee5ng. Sau ph\u1ea3n \u1ee9ng, n\u1ebfu \u0111\u01b0\u1eddng t\u00edn hi\u1ec7u c\u01b0\u1eddng \u0111\u1ed9 hu\u1ef3nh quang c\u1ee7a gen m\u1ee5c ti\u00eau v\u01b0\u1ee3t qua t\u00edn hi\u1ec7u n\u1ec1n th\u00ec x\u00e1c nh\u1eadn l\u00e0 c\u00f3 s\u1ef1 hi\u1ec7n di\u1ec7n c\u1ee7a t\u00e1c nh\u00e2n c\u1ea7n ki\u1ec3m tra v\u00e0 ng\u01b0\u1ee3c l\u1ea1i. Gi\u00e1 tr\u1ecb Ct s\u1edbm hay mu\u1ed9n ph\u1ea3n \u00e1nh s\u1ed1 l\u01b0\u1ee3ng b\u1ea3n DNA ban \u0111\u1ea7u gi\u00fap d\u1ef1 \u0111o\u00e1n t\u00ecnh tr\u1ea1ng b\u1ec7nh do t\u00e1c nh\u00e2n m\u1ee5c ti\u00eau g\u00e2y ra. <\/p>\n\n\n\n

Trong \u0111i\u1ec1u ki\u1ec7n l\u01b0\u1ee3ng m\u1eabu \u0111\u1ea7u v\u00e0o l\u00e0 nh\u01b0 nhau, m\u1eabu c\u00f3 Ct nh\u1ecf h\u01a1n th\u00ec s\u1ebd c\u00f3 h\u00e0m l\u01b0\u1ee3ng DNA cao h\u01a1n. Tuy nhi\u00ean trong th\u1ef1c t\u1ebf th\u00ec kh\u00f3 c\u00f3 th\u1ec3 kh\u1eb3ng \u0111\u1ecbnh l\u01b0\u1ee3ng m\u1eabu \u0111\u1ea7u v\u00e0o l\u00e0 nh\u01b0 nhau, v\u00ec v\u1eady c\u1ea7n ph\u1ea3i c\u00f3 gi\u00e1 tr\u1ecb Ct tham chi\u1ebfu c\u1ee7a gen ch\u1ee9ng n\u1ed9i (IC), vi\u1ec7c so s\u00e1nh s\u1ebd di\u1ec5n ra gi\u00e1n ti\u1ebfp th\u00f4ng qua vi\u1ec7c so s\u00e1nh v\u1edbi IC. Hi\u1ec7n nay tr\u00ean th\u1ecb tr\u01b0\u1eddng \u0111\u00e3 c\u00f3 nhi\u1ec1u b\u1ed9 kit qPCR \u0111\u1ecbnh t\u00ednh m\u1ed9t s\u1ed1 t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh truy\u1ec1n nhi\u1ec5m ph\u1ed5 bi\u1ebfn nh\u01b0 HBV, HCV, HPV,\u2026 cung c\u1ea5p gi\u1ea3i ph\u00e1p h\u1eefu \u00edch cho vi\u1ec7c ch\u1ea9n \u0111o\u00e1n b\u1ec7nh.<\/p>\n\n\n\n

5.2 Real-time PCR \u0111\u1ecbnh l\u01b0\u1ee3ng<\/strong><\/h3>\n\n\n\n

Kh\u00e1c v\u1edbi qPCR \u0111\u1ecbnh t\u00ednh ch\u1ec9 gi\u00fap x\u00e1c \u0111\u1ecbnh s\u1ef1 hi\u1ec7n di\u1ec7n c\u1ee7a t\u00e1c nh\u00e2n, k\u1ef9 thu\u1eadt qPCR \u0111\u1ecbnh l\u01b0\u1ee3ng s\u1ebd b\u1ed5 sung th\u00eam ch\u1ee9c n\u0103ng x\u00e1c \u0111\u1ecbnh ch\u00ednh x\u00e1c s\u1ed1 l\u01b0\u1ee3ng DNA m\u1ee5c ti\u00eau c\u00f3 trong m\u1eabu. K\u1ef9 thu\u1eadt n\u00e0y y\u00eau c\u1ea7u x\u00e2y d\u1ef1ng \u0111\u01b0\u1eddng chu\u1ea9n d\u1ef1a v\u00e0o gi\u00e1 tr\u1ecb Ct v\u00e0 d\u00e3y n\u1ed3ng \u0111\u1ed9 DNA \u0111\u00e3 bi\u1ebft tr\u01b0\u1edbc. N\u1ed3ng \u0111\u1ed9 DNA m\u1ee5c ti\u00eau s\u1ebd \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh th\u00f4ng qua \u0111\u01b0\u1eddng chu\u1ea9n, do v\u1eady c\u00e1c th\u00f4ng s\u1ed1 \u0111\u01b0\u1eddng chu\u1ea9n ph\u1ea3i \u0111\u1ea1t y\u00eau c\u1ea7u \u0111\u1ec3 ph\u1ea3n \u00e1nh \u0111\u1ed9 tin c\u1eady c\u0169ng nh\u01b0 ch\u1ea5t l\u01b0\u1ee3ng k\u1ebft qu\u1ea3 x\u00e9t nghi\u1ec7m c\u1ee7a qPCR \u0111\u1ecbnh l\u01b0\u1ee3ng.<\/p>\n\n\n\n

5.3. Quantitative multiplex reverse transcription PCR (Multiplex RT-qPCR)<\/strong><\/h3>\n\n\n\n

Hi\u1ec7n nay \u0111\u00e2y l\u00e0 k\u1ef9 thu\u1eadt kh\u1ea3 ph\u1ed5 bi\u1ebfn \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong nghi\u00ean c\u1ee9u v\u1ec1 ch\u1ee9c n\u0103ng gene, c\u1ee5 th\u1ec3 k\u1ef9 thu\u1eadt n\u00e0y cho ph\u00e9p \u0111o l\u01b0\u1eddng \u0111\u1ed3ng th\u1eddi s\u1ef1 hi\u1ec7n di\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng mRNA c\u1ee7a nhi\u1ec1u gen kh\u00e1c nhau trong m\u1ed9t m\u1eabu.<\/p>\n\n\n\n

N\u1ebfu ph\u00e2n c\u1eaft ngh\u0129a th\u00ec \u0111\u00e2y l\u00e0 k\u1ef9 thu\u1eadt k\u1ebft h\u1ee3p c\u1ee7a multiplex, reverse transcription v\u00e0 real-time PCR. Trong \u0111\u00f3, \u201cmultiplex\u201d d\u00f9ng \u0111\u1ec3 n\u00f3i \u0111\u1ebfn ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i t\u1eeb hai gen \u0111\u00edch tr\u1edf l\u00ean trong c\u00f9ng m\u1ed9t ph\u1ea3n \u1ee9ng (c\u00f3 th\u1ec3 tr\u00ean m\u1ed9t n\u1ec1n m\u1eabu hay nhi\u1ec1u n\u1ec1n m\u1eabu), c\u00f2n \u201creverse transcription\u201d (RT) l\u00e0 k\u1ef9 thu\u1eadt d\u00f9ng \u0111\u1ec3 chuy\u1ec3n \u0111\u1ed5i RNA s\u1ee3i khu\u00f4n th\u00e0nh s\u1ee3i DNA b\u1ed5 sung (complemantary DNA- cDNA) \u0111\u01b0\u1ee3c th\u1ef1c hi\u1ec7n b\u1edfi enzym phi\u00ean m\u00e3 ng\u01b0\u1ee3c (reverse transcriptase) v\u00e0 m\u1ed3i cho tr\u00ecnh t\u1ef1 RNA. <\/p>\n\n\n\n

Nh\u01b0 v\u1eady, nguy\u00ean t\u1eafc c\u1ee7a k\u1ef9 thu\u1eadt multiplex RT – qPCR l\u00e0 \u0111\u1ea7u ti\u00ean s\u1eed d\u1ee5ng c\u00e1c m\u1ed3i RT (nh\u1eefng \u0111o\u1ea1n m\u1ed3i ng\u1eafn g\u1eafn v\u1edbi s\u1ee3i khu\u00f4n mRNA) \u0111\u1ec3 kh\u1edfi \u0111\u1ed9ng qu\u00e1 tr\u00ecnh phi\u00ean m\u00e3 ng\u01b0\u1ee3c mRNA th\u00e0nh s\u1ee3i cDNA. Sau \u0111\u00f3, h\u1ed7n h\u1ee3p c\u00e1c c\u1eb7p m\u1ed3i kh\u00e1c nhau \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 nh\u00e2n b\u1ea3n c\u00e1c \u0111o\u1ea1n cDNA c\u1ee7a c\u00e1c gene c\u1ea7n x\u00e1c \u0111\u1ecbnh. Cu\u1ed1i c\u00f9ng, qPCR s\u1eed d\u1ee5ng probe gi\u00fap \u0111\u1ecbnh l\u01b0\u1ee3ng s\u1ed1 b\u1ea3n sao c\u1ee7a c\u00e1c \u0111o\u1ea1n cDNA \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh s\u1ed1 l\u01b0\u1ee3ng mRNA ban \u0111\u1ea7u trong m\u1eabu. <\/p>\n\n\n\n

K\u1ef9 thu\u1eadt k\u1ebft h\u1ee3p multiplex RT-qPCR l\u00e0 m\u1ed9t c\u00f4ng c\u1ee5 h\u1eefu \u00edch trong ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u1ed3ng th\u1eddi s\u1ef1 hi\u1ec7n di\u1ec7n c\u1ee7a nhi\u1ec1u mRNA m\u1ee5c ti\u00eau kh\u00e1c nhau trong m\u1ed9t m\u1eabu, gi\u00fap gi\u1ea3m th\u1eddi gian v\u00e0 chi ph\u00ed so v\u1edbi vi\u1ec7c th\u1ef1c hi\u1ec7n v\u1edbi t\u1eebng tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau kh\u00e1c nhau. <\/p>\n\n\n\n

Tuy nhi\u00ean, nh\u01b0\u1ee3c \u0111i\u1ec3m c\u1ee7a k\u1ef9 thu\u1eadt multiplex RT-qPCR \u0111\u00f3 l\u00e0 vi\u1ec7c thi\u1ebft k\u1ebf m\u1ed3i\/probe d\u00f9ng d\u1ec3 nh\u00e2n b\u1ea3n c\u00e1c tr\u00ecnh t\u1ef1 gene m\u1ee5c ti\u00eau kh\u00e1c nhau ph\u1ea3i \u0111\u1ea3m b\u1ea3o \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u, tr\u00e1nh hi\u1ec7n t\u01b0\u1ee3ng m\u1ed3i t\u01b0\u01a1ng t\u00e1c v\u1edbi nhau hay g\u1eafn kh\u00f4ng \u0111\u1eb7c hi\u1ec7u. B\u00ean c\u1ea1nh \u0111\u00f3, k\u1ef9 thu\u1eadt k\u1ebft h\u1ee3p n\u00e0y \u0111\u00f2i h\u1ecfi c\u00e1c th\u00f4ng s\u1ed1 c\u1ee7a qu\u00e1 tr\u00ecnh nh\u00e2n b\u1ea3n nh\u01b0 nhi\u1ec7t \u0111\u1ed9, th\u1eddi gian ph\u1ea3n \u1ee9ng, n\u1ed3ng \u0111\u1ed9 h\u00f3a ch\u1ea5t, \u2026 ph\u1ea3i \u0111\u01b0\u1ee3c kh\u1ea3o s\u00e1t \u0111\u1ec3 \u0111\u1ea3m b\u1ea3o t\u00ednh ch\u00ednh x\u00e1c c\u1ee7a k\u1ebft qu\u1ea3.<\/p>\n\n\n\n

Hi\u1ec7n nay, k\u1ef9 thu\u1eadt multiplex RT-qPCR \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng r\u1ed9ng r\u00e3i trong c\u00e1c b\u1ed9 kit \u0111\u1ec3 ch\u1ea9n \u0111o\u00e1n b\u1ec7nh. C\u00e1c b\u1ed9 kit n\u00e0y \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf \u0111\u1ec3 ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng \u0111\u1ed3ng th\u1eddi nhi\u1ec1u lo\u1ea1i t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh kh\u00e1c nhau trong m\u1ed9t hay nhi\u1ec1u n\u1ec1n m\u1eabu b\u1ec7nh ph\u1ea9m, gi\u00fap h\u1ed7 tr\u1ee3 b\u00e1c s\u0129 \u0111\u01b0a ra ph\u01b0\u01a1ng \u00e1n \u0111i\u1ec1u tr\u1ecb ph\u00f9 h\u1ee3p v\u00e0 ng\u0103n ng\u1eeba d\u1ecbch b\u1ec7nh l\u00e2y lan. <\/p>\n\n\n\n

M\u1ed9t s\u1ed1 b\u1ec7nh \u0111\u01b0\u1ee3c \u1ee9ng d\u1ee5ng k\u1ef9 thu\u1eadt trong c\u00e1c b\u1ed9 kit ch\u1ea9n nh\u01b0 kit ch\u1ea9n \u0111o\u00e1n virus c\u00fam lo\u1ea1i A\/B (Influenza virus type A\/B) \u1edf c\u00fam gia c\u1ea7m (TopSPEC\u00aeInfluenza virus A\/B RT-qPCR KIT<\/strong>), \u0111\u00e2y l\u00e0 b\u1ed9 kit x\u00e9t nghi\u1ec7m cho ph\u00e9p \u0111\u1ecbnh t\u00ednh ch\u00ednh x\u00e1c v\u00e0 ph\u00e1t hi\u1ec7n nhanh t\u00e1c nh\u00e2n g\u00e2y c\u00fam gia c\u1ea7m tr\u00ean nhi\u1ec1u n\u1ec1n m\u1eabu kh\u00e1c nhau. B\u1ed9 kit \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf k\u1ebft h\u1ee3p c\u00e1c b\u01b0\u1edbc trong m\u1ed9t l\u1ea7n l\u00e0m (onestep) n\u00ean t\u1ed5ng th\u1eddi gian trung b\u00ecnh cho vi\u1ec7c ph\u00e1t hi\u1ec7n t\u00e1c nh\u00e2n tr\u00ean to\u00e0n b\u1ed9 quy tr\u00ecnh l\u00e0 2 gi\u1edd, gi\u00fap ch\u1ea9n \u0111o\u00e1n s\u1edbm t\u00e1c nh\u00e2n v\u00e0 \u0111\u01b0a ra c\u00e1c bi\u1ec7n ph\u00e1p \u0111i\u1ec1u tr\u1ecb c\u0169ng nh\u01b0 c\u00e1ch ly c\u00e1 th\u1ec3 b\u1ec7nh s\u1edbm nh\u1ea5t. <\/p>\n\n\n\n

M\u1ed9t s\u1ed1 virus g\u00e2y b\u1ec7nh tr\u00ean th\u1ee7y s\u1ea3n nh\u01b0 YHV (Yellow head virus), TSV (Taura syndrrome inPenaeus vannamei) v\u00e0 IMNV (infectious myonecrosis virus) \u0111\u1ec1u c\u00f3 v\u1eadt ch\u1ea5t di truy\u1ec1n l\u00e0 mRNA n\u00ean c\u00f3 th\u1ec3 \u0111\u01b0\u1ee3c ph\u00e1t hi\u1ec7n c\u00f9ng l\u00fac trong m\u00f4i tr\u01b0\u1eddng nu\u00f4i tr\u1ed3ng khi s\u1eed d\u1ee5ng b\u1ed9 kit TopSPEC\u00ae YHV\/TSV\/IMNV Onestep RT-qPCR KIT.<\/p>\n\n\n\n

\u1ede ng\u01b0\u1eddi, k\u1ef9 thu\u1eadt n\u00e0y c\u0169ng \u0111\u00e3 \u0111\u01b0\u1ee3c \u1ee9ng d\u1ee5ng trong c\u00e1c b\u1ed9 kit ch\u1ea9n \u0111o\u00e1n SARS-CoV-2, \u0111i\u1ec3n h\u00ecnh l\u00e0 b\u1ed9 kit c\u1ee7a ScienCell (SCVMPD) \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf \u0111\u1ec3 ph\u00e1t hi\u1ec7n s\u1ef1 hi\u1ec7n di\u1ec7n c\u1ee7a SARS-CoV-2 trong c\u00e1c m\u1eabu b\u1ec7nh ph\u1ea9m \u0111\u01b0\u1eddng h\u00f4 h\u1ea5p v\u00e0 m\u1eabu huy\u1ebft thanh. Th\u00e0nh ph\u1ea7n ch\u1ee9a m\u1ed3i\/probe khu\u1ebfch \u0111\u1ea1i 3 tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau N1-FAM, N2-FAM v\u00e0 RP-HEX. Trong s\u1ed1 \u0111\u00f3, N1-FAM v\u00e0 N2-FAM nh\u1eafm m\u1ee5c ti\u00eau v\u00e0o hai v\u00f9ng tr\u00ean gen nucleocapsid (N) c\u1ee7a SARS-CoV-2. RP-HEX nh\u1eafm v\u00e0o exon 1 c\u1ee7a gen RPP30 \u1edf ng\u01b0\u1eddi v\u00e0 \u0111\u00f3ng vai tr\u00f2 ki\u1ec3m so\u00e1t \u0111\u1ec3 \u0111\u00e1nh gi\u00e1 ch\u1ea5t l\u01b0\u1ee3ng m\u1eabu.<\/p>\n\n\n\n

5.4. Droplet digital PCR (ddPCR)<\/strong><\/h3>\n\n\n\n

K\u1ef9 thu\u1eadt real-time PCR c\u00f3 th\u1ec3 \u0111\u1ecbnh l\u01b0\u1ee3ng tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau c\u00f3 trong m\u1eabu, tuy nhi\u00ean ch\u1ec9 \u1edf m\u1ee9c t\u01b0\u01a1ng \u0111\u1ed1i do h\u00e0m l\u01b0\u1ee3ng b\u1ea3n sao \u0111\u01b0\u1ee3c x\u00e1c \u0111\u1ecbnh ph\u1ee5 thu\u1ed9c v\u00e0o \u0111\u01b0\u1eddng chu\u1ea9n, m\u00e0 ch\u00ednh x\u00e1c l\u00e0 thao t\u00e1c ng\u01b0\u1eddi th\u1ef1c hi\u1ec7n, do v\u1eady k\u1ebft qu\u1ea3 c\u00f3 th\u1ec3 g\u00e2y sai l\u1ec7ch v\u00e0 kh\u00f4ng \u0111\u1ea3m b\u1ea3o \u0111\u1ed9 ch\u00ednh x\u00e1c cao. Droplet digital PCR (dddPCR) \u0111\u01b0\u1ee3c coi l\u00e0 k\u1ef9 thu\u1eadt thu\u1ed9c th\u1ebf h\u1ec7 m\u1edbi do \u0111\u01b0\u1ee3c c\u1ea3i ti\u1ebfn t\u1eeb real-time PCR cho ph\u00e9p \u0111\u1ecbnh l\u01b0\u1ee3ng ch\u00ednh x\u00e1c b\u1ea3n sao tr\u00ecnh t\u1ef1 m\u1ee5c ti\u00eau sau khi k\u1ebft th\u00fac qu\u00e1 tr\u00ecnh th\u1ef1c hi\u1ec7n.<\/p>\n\n\n\n

V\u1ec1 c\u01a1 b\u1ea3n, ddPCR c\u0169ng gi\u1ed1ng v\u1edbi qPCR v\u1ec1 th\u00e0nh ph\u1ea7n ph\u1ea3n \u1ee9ng bao g\u1ed3m mix, m\u1ed3i, probe v\u00e0 khu\u00f4n DNA nh\u01b0ng thay v\u00ec th\u1ec3 t\u00edch c\u1ee7a qPCR l\u00e0 20-25 \u00b5L\/ph\u1ea3n \u1ee9ng th\u00ec ddPCR s\u1ebd chia nh\u1ecf 20 \u00b5L th\u1ec3 t\u00edch ph\u1ea3n \u1ee9ng th\u00e0nh kho\u1ea3ng ch\u1ee5c ng\u00e0n vi gi\u1ecdt. M\u1ed7i vi gi\u1ecdt \u0111\u1ec1u c\u00f3 th\u1ec3 t\u00edch b\u1eb1ng nhau (kho\u1ea3ng 1nL\/ph\u1ea3n \u1ee9ng) v\u00e0 c\u00e1c vi gi\u1ecdt n\u00e0y s\u1ebd th\u1ef1c hi\u1ec7n m\u1ed9t ph\u1ea3n \u1ee9ng \u0111\u1ed9c l\u1eadp. Sau \u0111\u00f3 qu\u00e1 tr\u00ecnh nh\u00e2n b\u1ea3n \u0111\u01b0\u1ee3c ti\u1ebfn h\u00e0nh t\u01b0\u01a1ng t\u1ef1 qPCR, k\u1ebft qu\u1ea3 ghi nh\u1eadn c\u00f3 hay kh\u00f4ng c\u00f3 t\u00edn hi\u1ec7u hu\u1ef3nh quang c\u1ee7a vi gi\u1ecdt, ph\u1ea7n m\u1ec1m s\u1ebd \u00e1p d\u1ee5ng m\u00f4 h\u00ecnh ph\u00e2n ph\u1ed1i Poisson \u0111\u1ec3 t\u00ednh ra n\u1ed3ng \u0111\u1ed9 DNA m\u1ee5c ti\u00eau. <\/p>\n\n\n\n

Do \u0111\u00f3 k\u1ebft qu\u1ea3 \u0111\u1ecbnh l\u01b0\u1ee3ng c\u1ee7a ddPCR xem nh\u01b0 tuy\u1ec7t \u0111\u1ed1i v\u00ec kh\u00f4ng c\u1ea7n \u0111\u01b0\u1eddng cong chu\u1ea9n Ct nh\u01b0 qPCR. B\u00ean c\u1ea1nh \u0111\u00f3, \u0111i\u1ec3m m\u1ea1nh v\u01b0\u1ee3t tr\u1ed9i c\u1ee7a ddPCR so v\u1edbi qPCR \u0111\u00f3 l\u00e0 c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c s\u1ef1 kh\u00e1c nhau d\u00f9 l\u00e0 m\u1ed9t \u0111i\u1ec3m gi\u1eefa tr\u00ecnh t\u1ef1 c\u1ee7a ch\u1ee7ng hoang d\u1ea1i v\u00e0 ch\u1ee7ng \u0111\u1ed9t bi\u1ebfn v\u1edbi t\u1ef7 l\u1ec7 gene mang \u0111\u1ed9t bi\u1ebfn dao \u0111\u1ed9ng d\u01b0\u1edbi 10% . \u0110i\u1ec1u \u0111\u00f3 l\u00e0 nh\u1edd v\u00e0o k\u1ef9 thu\u1eadt chia vi gi\u1ecdt \u0111\u00e3 pha lo\u00e3ng DNA, d\u1eabn \u0111\u1ebfn gi\u1ea3m t\u00edn hi\u1ec7u n\u1ec1n l\u00e0m gi\u1ea3m m\u1ee9c \u0111\u1ed9 ch\u00eanh l\u1ec7ch tr\u00ecnh t\u1ef1 gi\u1eefa d\u1ea1ng hoang d\u1ea1i v\u00e0 \u0111\u1ed9t bi\u1ebfn, t\u1eeb \u0111\u00f3 l\u00e0m t\u0103ng kh\u1ea3 n\u0103ng ph\u00e1t hi\u1ec7n c\u00e1c \u0111\u1ed9t bi\u1ebfn c\u00f3 trong m\u1eabu. <\/p>\n\n\n\n

Nh\u01b0 v\u1eady c\u00f3 th\u1ec3 th\u1ea5y ddPCR mang nh\u1eefng \u01b0u \u0111i\u1ec3m v\u01b0\u1ee3t tr\u1ed9i v\u00e0 gi\u00fap kh\u1eafc ph\u1ee5c h\u1ea1n ch\u1ebf m\u00e0 qPCR g\u1eb7p ph\u1ea3i. Tuy nhi\u00ean, k\u1ef9 thu\u1eadt n\u00e0y hi\u1ec7n nay c\u00f3 chi ph\u00ed \u0111\u1ea7u t\u01b0 cao, th\u1eddi gian chu\u1ea9n b\u1ecb m\u1eabu l\u00e2u h\u01a1n v\u00e0 s\u1ed1 l\u01b0\u1ee3ng m\u1eabu x\u1eed l\u00fd c\u00f2n \u00edt. T\u00f9y v\u00e0o \u1ee9ng d\u1ee5ng m\u00e0 ch\u1ecdn k\u1ef9 thu\u1eadt ph\u00e2n t\u00edch ph\u00f9 h\u1ee3p,  v\u1edbi ddPCR, k\u1ef9 thu\u1eadt n\u00e0y r\u1ea5t ph\u00f9 h\u1ee3p cho ph\u00e1t hi\u1ec7n c\u00e1c gen mang \u0111\u1ed9t bi\u1ebfn hi\u1ebfm (\u0111\u1ed9t bi\u1ebfn \u0111i\u1ec3m hay SNP) \u0111\u01b0\u1ee3c \u1ee9ng d\u1ee5ng trong \u0111i\u1ec1u tr\u1ecb ung th\u01b0, ph\u00e1t hi\u1ec7n ch\u1ec9nh s\u1eeda b\u1ed9 gene hay  gi\u00e1m \u0111\u1ecbnh k\u1ebft qu\u1ea3 gi\u1ea3i tr\u00ecnh t\u1ef1 NGS cho nh\u1eefng \u0111\u1ed9t bi\u1ebfn \u1edf t\u1ea7n s\u1ed1 th\u1ea5p. <\/p>\n\n\n\n

Ng\u01b0\u1ee3c l\u1ea1i k\u1ef9 thu\u1eadt qPCR s\u1ebd ph\u00f9 h\u1ee3p v\u1edbi nh\u1eefng \u1ee9ng d\u1ee5ng \u0111\u01a1n gi\u1ea3n h\u01a1n nh\u01b0 ph\u00e1t hi\u1ec7n t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh ph\u1ed5 bi\u1ebfn (HBV, HCV, HPV hay HIV), \u0111\u00e2y l\u00e0 nh\u1eefng x\u00e9t nghi\u1ec7m n\u00e0y \u0111\u00f2i h\u1ecfi c\u00f4ng su\u1ea5t ph\u00e2n t\u00edch l\u1edbn, chi ph\u00ed th\u1ea5p v\u00e0 th\u1eddi gian gian cho k\u1ebft qu\u1ea3 nhanh ch\u00f3ng. Ngo\u00e0i ra c\u00e1c \u1ee9ng d\u1ee5ng kh\u00e1c nh\u01b0 nghi\u00ean c\u1ee9u bi\u1ec3u hi\u1ec7n gen, ph\u00e1t hi\u1ec7n siRNA, miRNA <\/a>hay lncRNA \u0111\u1ec1u n\u00ean \u01b0u ti\u00ean s\u1eed d\u1ee5ng qPCR thay v\u00ec k\u1ef9 thu\u1eadt ph\u1ee9c t\u1ea1p nh\u01b0 ddPCR.<\/p>\n\n\n\n

\"real-time
H\u00ecnh 9. S\u1ef1 kh\u00e1c nhau gi\u1eefa k\u1ef9 thu\u1eadt qPCR (A) v\u00e0 ddPCR (B)<\/em><\/figcaption><\/figure>\n\n\n\n

<\/p>\n\n\n\n

6. \u1ee8ng d\u1ee5ng c\u1ee7a k\u1ef9 thu\u1eadt real-time PCR<\/strong><\/h2>\n\n\n\n

K\u1ef9 thu\u1eadt real-time PCR l\u00e0 ph\u01b0\u01a1ng ph\u00e1p khu\u1ebfch \u0111\u1ea1i gen v\u00e0 \u0111\u1ed3ng th\u1eddi c\u00f3 th\u1ec3 \u0111\u1ecdc k\u1ebft qu\u1ea3 ngay t\u1ea1i th\u1eddi \u0111i\u1ec3m ph\u1ea3n \u1ee9ng x\u1ea3y ra. K\u1ebft qu\u1ea3 ph\u00e2n t\u00edch s\u1ebd \u0111\u01b0\u1ee3c th\u00f4ng qua t\u00edn hi\u1ec7u hu\u1ef3nh quang ph\u00e1t ra theo th\u1eddi gian t\u1ea1i m\u1ed7i chu k\u1ef3 c\u1ee7a ph\u1ea3n \u1ee9ng. K\u1ef9 thu\u1eadt n\u00e0y \u0111\u01b0\u1ee3c coi l\u00e0 b\u01b0\u1edbc ti\u1ebfn l\u1edbn c\u1ee7a PCR v\u00e0 \u0111\u1eb7c bi\u1ec7t \u0111\u01b0\u1ee3c \u1ee9ng d\u1ee5ng nhi\u1ec1u trong ch\u1ea9n \u0111o\u00e1n b\u1ec7nh, x\u00e1c \u0111\u1ecbnh c\u00e1c sinh v\u1eadt bi\u1ebfn \u0111\u1ed5i gen v\u00e0 ki\u1ec3u gen c\u00e1c t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh. B\u00ean c\u1ea1nh \u0111\u00f3, trong nghi\u00ean c\u1ee9u, real-time PCR c\u00f2n ph\u00e1t hi\u1ec7n s\u1ef1 bi\u1ebfn \u0111\u1ed5i trong bi\u1ec3u hi\u1ec7n c\u1ee7a m\u1ed9t gen qua th\u00f4ng qua \u0111\u1ecbnh l\u01b0\u1ee3ng s\u1ed1 phi\u00ean m\u00e3 c\u1ee7a gen.<\/p>\n\n\n\n

6.1 Ch\u1ea9n \u0111o\u00e1n b\u1ec7nh truy\u1ec1n nhi\u1ec5m tr\u00ean ng\u01b0\u1eddi<\/strong><\/h3>\n\n\n\n

\u0110\u1ed1i v\u1edbi b\u1ec7nh truy\u1ec1n nhi\u1ec5m, vi\u1ec7c x\u00e1c \u0111\u1ecbnh t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh v\u00e0 c\u00e1ch ly c\u00e1c c\u00e1 th\u1ec3 b\u1ec7nh l\u00e0 vi\u1ec7c h\u1ebft s\u1ee9c quan tr\u1ecdng trong ki\u1ec3m so\u00e1t d\u1ecbch b\u1ec7nh. C\u00e1c ph\u01b0\u01a1ng ph\u00e1p ch\u1ea9n \u0111o\u00e1n c\u00e1c t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh th\u01b0\u1eddng d\u1ef1a v\u00e0o tri\u1ec7u ch\u1ee9ng l\u00e2m s\u00e0ng hay ki\u1ec3m tra h\u00ecnh \u1ea3nh. Tr\u01b0\u1eddng h\u1ee3p tri\u1ec7u ch\u1ee9ng hay h\u00ecnh \u1ea3nh ch\u01b0a r\u00f5 r\u00e0ng ho\u1eb7c kh\u00f4ng \u0111\u1eb7c tr\u01b0ng th\u00ec x\u00e9t nghi\u1ec7m real-time PCR \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 x\u00e1c \u0111\u1ecbnh ch\u00ednh x\u00e1c s\u1ef1 t\u1ed3n t\u1ea1i v\u00e0 t\u1ea3i l\u01b0\u1ee3ng c\u1ee7a t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh, t\u1eeb \u0111\u00f3 c\u00f3 th\u1ec3 ng\u0103n ng\u1eeba l\u00e2y lan d\u1ecbch b\u1ec7nh v\u00e0 \u0111i\u1ec1u tr\u1ecb hi\u1ec7u qu\u1ea3 cho b\u1ec7nh nh\u00e2n.<\/p>\n\n\n\n

\u0110\u1ec3 \u0111\u00e1p \u1ee9ng y\u00eau c\u1ea7u vi\u1ec7c x\u00e9t nghi\u1ec7m qPCR c\u1ea7n k\u1ebft qu\u1ea3 ch\u00ednh x\u00e1c v\u00e0 nhanh ch\u00f3ng, c\u00e1c nh\u00e0 nghi\u00ean c\u1ee9u k\u1ebft h\u1ee3p v\u1edbi c\u00e1c nh\u00e0 s\u1ea3n xu\u1ea5t s\u1ea3n ph\u1ea9m y sinh \u0111\u00e3 cho ra c\u00e1c b\u1ed9 kit s\u1eb5n s\u00e0ng cho ch\u1ea9n \u0111o\u00e1n c\u00e1c t\u00e1c nh\u00e2n ph\u1ed5 bi\u1ebfn nh\u01b0 HCV, HBV, HPV, b\u1ec7nh l\u1eadu,… \u0110\u1ed1i v\u1edbi kit s\u1eed d\u1ee5ng SYBR Green ch\u1ec9 ph\u00e1t hi\u1ec7n \u0111\u01b0\u1ee3c \u0111\u01a1n t\u00e1c nh\u00e2n (singleplex) do t\u00ednh kh\u00f4ng \u0111\u1eb7c hi\u1ec7u v\u1ec1 tr\u00ecnh t\u1ef1 c\u1ee7a ch\u1ea5t hu\u1ef3nh quang d\u1ea1ng n\u00e0y. V\u1edbi c\u00e1c b\u1ed9 kit s\u1eed d\u1ee5ng probe c\u00f3 kh\u1ea3 n\u0103ng ph\u00e1t hi\u1ec7n nhi\u1ec1u \u0111o\u1ea1n gen hay nhi\u1ec1u t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh s\u1ebd gi\u00fap t\u1ed1i \u01b0u ho\u00e1 \u0111\u1ed9 nh\u1ea1y c\u0169ng nh\u01b0 \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u cho x\u00e9t nghi\u1ec7m. <\/p>\n\n\n\n

V\u00ed d\u1ee5, vi\u00eam gan B (Hepatis B Virus – HBV) l\u00e0 vi r\u00fat thu\u1ed9c h\u1ecd Hepadnaviridae c\u00f3 c\u1ea5u tr\u00fac DNA m\u1ea1ch \u0111\u00f4i, \u0111\u00e2y l\u00e0 b\u1ec7nh truy\u1ec1n nhi\u1ec5m \u1ea3nh h\u01b0\u1edfng l\u1edbn \u0111\u1ebfn ho\u1ea1t \u0111\u1ed9ng c\u1ee7a gan, c\u00f3 th\u1ec3 g\u00e2y nhi\u1ec5m tr\u00f9ng gan th\u1eadm ch\u00ed \u0111e d\u1ecda \u0111\u1ebfn t\u00ednh m\u1ea1ng. V\u00ec v\u1eady, c\u00f4ng t\u00e1c ph\u00e1t hi\u1ec7n v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng ch\u00ednh x\u00e1c n\u1ed3ng \u0111\u1ed9 HBV trong m\u00e1u r\u1ea5t quan tr\u1ecdng, gi\u00fap vi\u1ec7c \u0111i\u1ec1u tr\u1ecb cho b\u1ec7nh nh\u00e2n d\u1ec5 d\u00e0ng h\u01a1n. B\u1ed9 kit l\u01b0u h\u00e0nh hi\u1ec7n nay c\u0169ng \u0111\u00e3 \u0111\u01b0\u1ee3c t\u1ed1i \u01b0u h\u00f3a \u0111\u1ec3 t\u0103ng kh\u1ea3 n\u0103ng ph\u00e1t hi\u1ec7n to\u00e0n b\u1ed9 genotype HBV t\u1ea1i v\u00f9ng 37S protein gen v\u1edbi \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u v\u00e0 \u0111\u1ed9 nh\u1ea1y cao. <\/p>\n\n\n\n

B\u00ean c\u1ea1nh \u0111\u00f3, c\u00e1c b\u1ed9 kit HBV<\/a><\/strong> ngo\u00e0i \u0111\u1ecbnh t\u00ednh c\u00f2n cung c\u1ea5p kh\u1ea3 n\u0103ng \u0111\u1ecbnh l\u01b0\u1ee3ng ch\u00ednh x\u00e1c n\u1ed3ng \u0111\u1ed9 HBV trong m\u1eabu huy\u1ebft thanh\/ huy\u1ebft t\u01b0\u01a1ng \u1edf ng\u01b0\u1eddi. Trong b\u1ed9 kit cung c\u1ea5p c\u00e1c \u1ed1ng ch\u1ee9a c\u00e1c m\u1eabu \u0111\u00e3 chu\u1ea9n n\u1ed3ng \u0111\u1ed9, \u0111\u01b0\u1eddng chu\u1ea9n \u0111\u01b0\u1ee3c x\u00e2y d\u1ef1ng d\u1ef1a tr\u00ean 5 \u0111i\u1ec3m (\u1ed1ng m\u1eabu) mang l\u1ea1i k\u1ebft qu\u1ea3 \u0111\u1ecbnh l\u01b0\u1ee3ng ch\u00ednh x\u00e1c, k\u1ebft h\u1ee3p v\u1edbi c\u00e1c h\u1ec7 th\u1ed1ng \u0111\u1ed1i ch\u1ee9ng \u00e2m, \u0111\u1ed1i ch\u1ee9ng d\u01b0\u01a1ng, \u0111\u1ed1i ch\u1ee9ng n\u1ed9i <\/a><\/strong>nh\u1eb1m ph\u00e1t hi\u1ec7n k\u1ecbp th\u1eddi c\u00e1c k\u1ebft qu\u1ea3 b\u1ea5t th\u01b0\u1eddng, gi\u00fap vi\u1ec7c theo d\u00f5i v\u00e0 \u0111i\u1ec1u tr\u1ecb b\u1ec7nh hi\u1ec7u qu\u1ea3 h\u01a1n.<\/p>\n\n\n\n

Trong khi \u0111\u00f3, \u0111\u1ed1i v\u1edbi virus vi\u00eam gan C (Hepatis C Virus – HCV)<\/a><\/strong> l\u00e0 k\u1ebb gi\u1ebft ng\u01b0\u1eddi th\u1ea7m l\u1eb7ng, v\u1edbi t\u1ef7 l\u1ec7 bi\u1ebfn ch\u1ee9ng x\u01a1 gan, ung th\u01b0 gan cao h\u01a1n r\u1ea5t nhi\u1ec1u so v\u1edbi HBV. C\u1ea5u tr\u00fac gen c\u1ee7a HCV c\u00f3 d\u1ea1ng m\u1ea1ch \u0111\u01a1n RNA, do v\u1eady c\u1ea7n chuy\u1ec3n \u0111\u1ed5i th\u00e0nh cDNA \u0111\u1ec3 l\u00e0m khu\u00f4n m\u1eabu cho ph\u1ea3n \u1ee9ng khu\u1ebfch \u0111\u1ea1i. Tr\u00ean th\u1ecb tr\u01b0\u1eddng hi\u1ec7n nay, c\u00e1c b\u1ed9 kit c\u0169ng cung c\u1ea5p c\u00e1c gi\u1ea3i ph\u00e1p one-step hay two-step t\u00f9y v\u00e0o nhu c\u1ea7u s\u1eed d\u1ee5ng v\u1edbi \u01b0u nh\u01b0\u1ee3c \u0111i\u1ec3m ri\u00eang. B\u1ed9 m\u1ed3i – probe c\u0169ng \u0111\u01b0\u1ee3c thi\u1ebft k\u1ebf g\u1eafn \u0111\u1eb7c hi\u1ec7u c\u00f3 kh\u1ea3 n\u0103ng ph\u00e1t hi\u1ec7n to\u00e0n b\u1ed9 genotype HCV t\u1ea1i v\u00f9ng HCV73 5\u2032 UTR. <\/p>\n\n\n\n

Ngo\u00e0i kh\u1ea3 n\u0103ng ph\u00e1t hi\u1ec7n HCV nhanh trong m\u1eabu b\u1ec7nh ph\u1ea9m th\u00ec m\u1ed9t s\u1ed1 kit c\u0169ng cung c\u1ea5p \u0111\u01b0\u1eddng chu\u1ea9n x\u00e1c \u0111\u1ecbnh t\u1ea3i l\u01b0\u1ee3ng HCV gi\u00fap \u0111i\u1ec1u tr\u1ecb t\u1ed1t h\u01a1n. Hay c\u00e1c kit x\u00e9t nghi\u1ec7m SARS-COV-2 l\u01b0u h\u00e0nh tr\u00ean th\u1ecb tr\u01b0\u1eddng hi\u1ec7n nay c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n c\u00e1c gen \u0111\u1eb7c tr\u01b0ng c\u1ee7a vi r\u00fat bao g\u1ed3m gen N, gen E v\u00e0 RdRp. M\u1ed9t c\u00f4ng b\u1ed1 tr\u00ean T\u1ea1p ch\u00ed Nature g\u1ea7n \u0111\u00e2y \u0111\u00e3 c\u1ea3i ti\u1ebfn k\u1ef9 thu\u1eadt multiplex qPCR kh\u00f4ng ch\u1ec9 ph\u00e1t hi\u1ec7n c\u00e1c gen tr\u00ean vi r\u00fat m\u00e0 c\u00f2n gen RP \u1edf ng\u01b0\u1eddi \u0111\u1ec3 gia t\u0103ng hi\u1ec7u qu\u1ea3 x\u00e9t nghi\u1ec7m [Tombuloglu et al., 2022].<\/p>\n\n\n\n

6.2. Ch\u1ea9n \u0111o\u00e1n b\u1ec7nh tr\u00ean th\u00fa y, th\u1ee7y s\u1ea3n<\/strong><\/h3>\n\n\n\n

C\u00e1c b\u1ec7nh l\u00e2y truy\u1ec1n t\u1eeb \u0111\u1ed9ng v\u1eadt sang ng\u01b0\u1eddi \u0111ang c\u00f3 chi\u1ec1u h\u01b0\u1edbng ng\u00e0y c\u00e0ng gia t\u0103ng. Th\u00f4ng th\u01b0\u1eddng c\u00e1c trang tr\u1ea1i v\u1eabn kh\u00e1 l\u01a1 l\u00e0 trong vi\u1ec7c ph\u00f2ng ng\u1eeba d\u1ecbch b\u1ec7nh, khi c\u00e1c \u1ed5 d\u1ecbch b\u00f9ng ph\u00e1t th\u00ec m\u1edbi c\u00f3 h\u00e0nh \u0111\u1ed9ng kh\u1eafc ph\u1ee5c h\u1eadu qu\u1ea3 v\u00e0 quan t\u00e2m x\u00e2y d\u1ef1ng bi\u1ec7n ph\u00e1p ng\u0103n ng\u1eeba m\u1ea7m b\u1ec7nh x\u00e2m nh\u1eadp. <\/p>\n\n\n\n

\u0110\u1ec3 ch\u1ea9n \u0111o\u00e1n b\u1ec7nh tr\u00ean v\u1eadt nu\u00f4i, ng\u01b0\u1eddi d\u00e2n hay ki\u1ec3m tra b\u1eb1ng c\u1ea3m quan nh\u01b0 c\u00e1c tri\u1ec7u ch\u1ee9ng tr\u00ean da, nhi\u1ec7t \u0111\u1ed9 c\u01a1 th\u1ec3, theo d\u00f5i s\u1ee9c \u0103n v\u00e0 ho\u1ea1t \u0111\u1ed9ng c\u1ee7a v\u1eadt nu\u00f4i,\u2026 Tuy nhi\u00ean c\u00e1c ph\u01b0\u01a1ng ph\u00e1p n\u00e0y th\u01b0\u1eddng kh\u00f4ng ch\u00ednh x\u00e1c v\u00e0 kh\u00f3 ph\u00e1t hi\u1ec7n s\u1edbm c\u00e1c m\u1ea7m b\u1ec7nh. \u0110\u1ec3 h\u1ea1n ch\u1ebf thi\u1ec7t h\u1ea1i, vi\u1ec7c t\u0103ng kh\u1ea3 n\u0103ng \u1ee9ng d\u1ee5ng c\u00f4ng ngh\u1ec7 cao trong ch\u1ea9n \u0111o\u00e1n, gi\u00e1m s\u00e1t d\u1ecbch b\u1ec7nh tr\u00ean \u0111\u1ed9ng v\u1eadt l\u00e0 r\u1ea5t c\u1ea7n thi\u1ebft. <\/p>\n\n\n\n

M\u1ed9t s\u1ed1 ph\u01b0\u01a1ng ph\u00e1p \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng \u0111\u1ec3 ph\u00e1t hi\u1ec7n s\u1ef1 hi\u1ec7n di\u1ec7n c\u1ee7a vi khu\u1ea9n, vi r\u00fat g\u00e2y b\u1ec7nh tr\u00ean v\u1eadt nu\u00f4i nh\u01b0: ph\u00e2n l\u1eadp nu\u00f4i c\u1ea5y vi sinh truy\u1ec1n th\u1ed1ng, th\u1eed nghi\u1ec7m sinh h\u00f3a truy\u1ec1n th\u1ed1ng, kit test nhanh, Elisa,\u2026 \u0110i\u1ec3m chung c\u1ee7a c\u00e1c ph\u01b0\u01a1ng ph\u00e1p n\u00e0y \u0111\u1ec1u c\u00f3 \u0111\u1ed9 nh\u1ea1y v\u00e0 \u0111\u1ed9 \u0111\u1eb7c hi\u1ec7u kh\u00f4ng cao ho\u1eb7c th\u1eddi gian th\u1eed nghi\u1ec7m l\u00e2u, kh\u00f4ng ph\u00f9 h\u1ee3p v\u1edbi c\u00e1c \u0111\u01a1n v\u1ecb s\u1ea3n xu\u1ea5t c\u1ea7n k\u1ebft qu\u1ea3 nhanh v\u00e0 c\u00f3 \u0111\u1ed9 tin c\u1eady cao. <\/p>\n\n\n\n

\u0110\u1ec3 \u0111\u00e1p \u1ee9ng nh\u1eefng nhu c\u1ea7u tr\u00ean, x\u00e9t nghi\u1ec7m real-time PCR l\u00e0 m\u1ed9t trong nh\u1eefng c\u00f4ng c\u1ee5 ch\u1ea9n \u0111o\u00e1n ph\u1ed5 bi\u1ebfn nh\u1ea5t ng\u00e0y nay \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng trong th\u00fa y, th\u1ee7y s\u1ea3n. K\u1ef9 thu\u1eadt n\u00e0y kh\u00f4ng ch\u1ec9 \u0111\u01b0\u1ee3c s\u1eed d\u1ee5ng cho m\u1ee5c \u0111\u00edch ch\u1ea9n \u0111o\u00e1n m\u00e0 c\u00f2n l\u00e0 ph\u01b0\u01a1ng ti\u1ec7n \u0111\u1ec3 c\u1ea3i thi\u1ec7n an to\u00e0n sinh h\u1ecdc th\u00f4ng qua gi\u00e1m s\u00e1t v\u00e0 theo d\u00f5i d\u1ecbch b\u1ec7nh. <\/p>\n\n\n\n

V\u1edbi s\u1ef1 ph\u00e1t tri\u1ec3n k\u1ef9 thu\u1eadt qPCR, c\u00e1c d\u00f2ng kit ph\u00e1t hi\u1ec7n b\u1ec7nh ng\u00e0y c\u00e0ng \u0111a d\u1ea1ng tr\u00ean v\u1eadt nu\u00f4i \u0111\u1ec3 ph\u00e1t hi\u1ec7n c\u00e1c v\u1eadt li\u1ec7u di truy\u1ec1n l\u00e0 DNA ho\u1eb7c RNA c\u1ee7a c\u00e1c t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh (nh\u01b0 vi khu\u1ea9n, vi r\u00fat, vi b\u00e0o t\u1eed), th\u00f4ng qua h\u1ec7 m\u1ed3i \u2013 m\u1eabu d\u00f2 \u0111\u1eb7c hi\u1ec7u c\u00f3 \u0111\u1ed9 ch\u00ednh x\u00e1c r\u1ea5t cao v\u00e0 k\u1ebft qu\u1ea3 c\u00f3 th\u1ec3 \u0111\u1ecdc tr\u1ef1c ti\u1ebfp tr\u00ean m\u00e1y t\u00ednh trong th\u1eddi gian ng\u1eafn. C\u00e1c d\u00f2ng kit ph\u1ed5 bi\u1ebfn tr\u00ean \u0111a d\u1ea1ng b\u1ec7nh nh\u01b0: B\u1ec7nh t\u1ea3 l\u1ee3n ch\u00e2u Phi do African Swine Fever Virus (ASFV), l\u1edf m\u1ed3m long m\u00f3ng do Foot and Mouth Disease Virus (FMDV), d\u1ecbch t\u1ea3 heo c\u1ed5 \u0111i\u1ec3n do Classical Swine Fever Virus (CSFV), \u2026<\/p>\n\n\n\n

B\u00ean c\u1ea1nh \u0111\u00f3, nu\u00f4i tr\u1ed3ng th\u1ee7y s\u1ea3n c\u0169ng l\u00e0 m\u1ed9t trong nh\u1eefng ng\u00e0nh c\u00f4ng nghi\u1ec7p ph\u00e1t tri\u1ec3n nhanh nh\u1ea5t \u1edf n\u01b0\u1edbc ta. Vi\u1ec7c th\u00e2m canh nu\u00f4i t\u00f4m m\u1eadt \u0111\u1ed9 cao ng\u00e0y c\u00e0ng nhi\u1ec1u, k\u00e9o theo nhi\u1ec1u th\u00e1ch th\u1ee9c v\u00e0 r\u1ee7i ro. C\u00e1c \u0111\u1ea1i d\u1ecbch tr\u00ean t\u00f4m nh\u01b0 \u0111\u1ed1m tr\u1eafng (WSSV), H\u1ed9i ch\u1ee9ng t\u00f4m ch\u1ebft s\u1edbm hay b\u1ec7nh ho\u1ea1i t\u1eed gan t\u1ee5y c\u1ea5p (EMS\/AHPND), b\u1ec7nh \u0111\u1ea7u v\u00e0ng YHV, ho\u1ea1i t\u1eed c\u01a1 quan t\u1ea1o m\u00e1u v\u00e0 bi\u1ec3u m\u00f4 IHHNV,\u2026 t\u1eebng g\u00e2y t\u1ed5n th\u1ea5t h\u00e0ng tr\u0103m ng\u00e0n t\u1ea5n t\u00f4m thi\u1ec7t h\u1ea1i h\u00e0ng t\u1ef7 USD m\u1ed7i n\u0103m cho ng\u00e0nh t\u00f4m to\u00e0n c\u1ea7u. <\/p>\n\n\n\n

Th\u00f4ng th\u01b0\u1eddng \u0111\u1ec3 ch\u1ea9n \u0111o\u00e1n b\u1ec7nh cho t\u00f4m, ng\u01b0\u1eddi d\u00e2n hay s\u1eed d\u1ee5ng kinh nghi\u1ec7m nu\u00f4i l\u00e2u n\u0103m nh\u01b0 ki\u1ec3m tra b\u1eb1ng c\u1ea3m quan c\u00e1c tri\u1ec7u ch\u1ee9ng tr\u00ean mang, v\u1ea3y, hi\u1ec7n t\u01b0\u1ee3ng c\u1ee7a n\u01b0\u1edbc ao nu\u00f4i, t\u00ecnh tr\u1ea1ng v\u1eadt nu\u00f4i, \u2026 Tuy nhi\u00ean, nh\u1eefng ph\u01b0\u01a1ng ph\u00e1p n\u00e0y th\u01b0\u1eddng kh\u00f4ng ch\u00ednh x\u00e1c v\u00e0 kh\u00f3 c\u00f3 th\u1ec3 ph\u00e1t hi\u1ec7n s\u1edbm \u0111\u01b0\u1ee3c m\u1ea7m b\u1ec7nh. Do \u0111\u00f3, vi\u1ec7c ph\u00f2ng ng\u1eeba d\u1ecbch b\u1ec7nh tr\u00ean th\u1ee7y s\u1ea3n c\u00f3 \u00fd ngh\u0129a quan tr\u1ecdng, gi\u1ea3m thi\u1ec3u thi\u1ec7t h\u1ea1i cho ng\u01b0\u1eddi nu\u00f4i, \u0111\u1ecbnh h\u01b0\u1edbng c\u00e1ch x\u1eed l\u00fd nh\u1eb1m gi\u1ea3m thi\u1ec3u r\u1ee7i ro \u0111\u1ebfn m\u1ee9c th\u1ea5p nh\u1ea5t. <\/p>\n\n\n\n

Nhi\u1ec1u nh\u00e0 s\u1ea3n xu\u1ea5t \u0111\u00e3 cung c\u1ea5p nhi\u1ec1u d\u00f2ng s\u1ea3n ph\u1ea9m kit ch\u1ea9n \u0111o\u00e1n \u0111a d\u1ea1ng t\u00e1c nh\u00e2n g\u00e2y b\u1ec7nh tr\u00ean t\u00f4m, s\u1eed d\u1ee5ng cho c\u00e1c x\u00e9t nghi\u1ec7m tr\u01b0\u1edbc v\u00e0 trong su\u1ed1t qu\u00e1 tr\u00ecnh nu\u00f4i nh\u1eb1m t\u1ea7m so\u00e1t nguy c\u01a1 nhi\u1ec5m b\u1ec7nh, ph\u00e1t hi\u1ec7n s\u1edbm \u0111\u01b0\u1ee3c m\u1ea7m b\u1ec7nh trong m\u00f4i tr\u01b0\u1eddng nu\u00f4i. \u0110\u1eb7c bi\u1ec7t l\u00e0 nh\u1eefng b\u1ec7nh c\u00f3 nguy c\u01a1 t\u1eed vong cao ho\u1eb7c n\u1eb1m trong y\u00eau c\u1ea7u x\u00e9t nghi\u1ec7m \u0111\u1ec3 \u0111\u1ee7 \u0111i\u1ec1u ki\u1ec7n xu\u1ea5t nh\u1eadp kh\u1ea9u qua c\u00e1c th\u1ecb tr\u01b0\u1eddng n\u01b0\u1edbc ngo\u00e0i.<\/p>\n\n\n\n

6.3 X\u00e1c \u0111\u1ecbnh th\u1ef1c ph\u1ea9m GMO<\/strong><\/h3>\n\n\n\n

Th\u1ef1c ph\u1ea9m bi\u1ebfn \u0111\u1ed5i gen hay c\u00f2n g\u1ecdi l\u00e0 th\u1ef1c ph\u1ea9m GM (Genetically modified) \u0111\u01b0\u1ee3c d\u00f9ng \u0111\u1ec3 ch\u1ec9 c\u00e1c lo\u1ea1i th\u1ef1c ph\u1ea9m c\u00f3 th\u00e0nh ph\u1ea7n t\u1eeb c\u00e2y tr\u1ed3ng hay \u0111\u1ed9ng v\u1eadt bi\u1ebfn \u0111\u1ed5i gen. S\u1ef1 an to\u00e0n v\u1ec1 th\u1ef1c ph\u1ea9m GM hi\u1ec7n nay v\u1eabn l\u00e0 ch\u1ee7 \u0111\u1ec1 n\u00f3ng g\u00e2y tranh c\u00e3i th\u1eddi gian qua, m\u1eb7c d\u00f9 t\u1ed5 ch\u1ee9c WHO v\u00e0 FAO ch\u01b0a c\u00f3 b\u00e1o c\u00e1o n\u00e0o \u0111\u1ec1 c\u1eadp \u0111\u1ebfn nh\u1eefng t\u00e1c \u0111\u1ed9ng nguy h\u1ea1i \u1edf m\u1ee9c b\u00e1o \u0111\u1ed9ng c\u1ee7a GMO cho ng\u01b0\u1eddi s\u1eed d\u1ee5ng nh\u01b0 \u0111\u1ed9t bi\u1ebfn hay g\u00e2y t\u1eed vong. <\/p>\n\n\n\n

Tuy nhi\u00ean, c\u00e1c nh\u00e0 nghi\u00ean c\u1ee9u khoa h\u1ecdc c\u0169ng cho th\u1ea5y lo\u1ea1i th\u1ef1c ph\u1ea9m n\u00e0y c\u00f3 kh\u1ea3 n\u0103ng g\u00e2y d\u1ecb \u1ee9ng, g\u00e2y kh\u00e1ng kh\u00e1ng sinh hay r\u1ed1i lo\u1ea1n qu\u00e1 tr\u00ecnh chuy\u1ec3n h\u00f3a. Lu\u1eadt ghi nh\u00e3n b\u1eaft bu\u1ed9c th\u01b0\u1eddng c\u00f3 hi\u1ec7u l\u1ef1c d\u1ef1a tr\u00ean t\u1ef7 l\u1ec7 GMO c\u1ee7a b\u1ea5t k\u1ef3 th\u00e0nh ph\u1ea7n c\u1ee5 th\u1ec3 n\u00e0o trong m\u1ed9t s\u1ea3n ph\u1ea9m th\u1ef1c ph\u1ea9m, m\u1eb7c d\u00f9 m\u1ed9t s\u1ed1 quy \u0111\u1ecbnh d\u1ef1a tr\u00ean t\u1ef7 l\u1ec7 ph\u1ea7n tr\u0103m tr\u00ean to\u00e0n b\u1ed9 m\u1eb7t h\u00e0ng th\u1ef1c ph\u1ea9m. C\u00e1c quy t\u1eafc h\u1ea1n ch\u1ebf nh\u1ea5t, nh\u01b0 Li\u00ean minh ch\u00e2u \u00c2u, \u1ea2 R\u1eadp X\u00ea-\u00fat, Th\u1ed5 Nh\u0129 K\u1ef3 v\u00e0 \u00dac, c\u00f3 ng\u01b0\u1ee1ng 0,9%. <\/p>\n\n\n\n

Nh\u1eefng n\u01b0\u1edbc kh\u00e1c cho ph\u00e9p bao g\u1ed3m GMO \u1edf m\u1ee9c \u0111\u1ed9 cao h\u01a1n, v\u1edbi H\u00e0n Qu\u1ed1c l\u00e0 3% v\u00e0 Nh\u1eadt B\u1ea3n l\u00e0 5%. \u00dac v\u00e0 New Zealand y\u00eau c\u1ea7u c\u00e1c th\u00e0nh ph\u1ea7n GMO ph\u1ea3i \u0111\u01b0\u1ee3c ghi ch\u00fa trong b\u1ea3ng th\u00e0nh ph\u1ea7n, tr\u1eeb khi to\u00e0n b\u1ed9 m\u1eb7t h\u00e0ng l\u00e0 bi\u1ebfn \u0111\u1ed5i gen. Trong tr\u01b0\u1eddng h\u1ee3p \u0111\u00f3, th\u00f4ng tin ph\u1ea3i \u0111\u01b0\u1ee3c bao g\u1ed3m b\u00ean c\u1ea1nh t\u00ean, theo C\u01a1 quan Ti\u00eau chu\u1ea9n Th\u1ef1c ph\u1ea9m Australia, New Zealand. H\u00e0n Qu\u1ed1c c\u00f3 ng\u01b0\u1ee1ng d\u00e1n nh\u00e3n GMO cao h\u01a1n, v\u1edbi b\u1ea5t k\u1ef3 th\u1ee9 g\u00ec d\u01b0\u1edbi 3% \u0111\u01b0\u1ee3c coi l\u00e0 kh\u00f4ng c\u1ed1 \u00fd. <\/p>\n\n\n\n

Do v\u1eady, ki\u1ec3m nghi\u1ec7m th\u1ef1c ph\u1ea9m bi\u1ebfn \u0111\u1ed5i gen l\u00e0 \u0111i\u1ec1u c\u1ea7n thi\u1ebft \u0111\u1ec3 x\u00e1c nh\u1eadn v\u00e0 \u0111\u1ecbnh l\u01b0\u1ee3ng sinh v\u1eadt bi\u1ebfn \u0111\u1ed5i gen trong c\u00e1c m\u1eabu th\u1ef1c ph\u1ea9m ho\u1eb7c th\u1ee9c \u0103n ch\u0103n nu\u00f4i. K\u1ef9 thu\u1eadt real-time PCR th\u01b0\u1eddng \u0111\u01b0\u1ee3c \u1ee9ng d\u1ee5ng hi\u1ec7u qu\u1ea3 trong d\u00e1n nh\u00e3n v\u00e0 x\u00e1c \u0111\u1ecbnh h\u00e0m l\u01b0\u1ee3ng GMO. M\u1ed7i t\u1ed5 ch\u1ee9c hay qu\u1ed1c gia s\u1ebd quy \u0111\u1ecbnh ph\u1ea7n tr\u0103m GMO \u0111\u1ec3 d\u00e1n nh\u00e3n cho m\u1eabu, do \u0111\u00f3 \u0111\u1ec3 gi\u00fap doanh nghi\u1ec7p th\u1ef1c ph\u1ea9m c\u00f3 th\u1ec3 xu\u1ea5t kh\u1ea9u v\u00e0o c\u00e1c th\u1ecb tr\u01b0\u1eddng tr\u00ean th\u1ebf gi\u1edbi, vi\u1ec7c ph\u00e1t hi\u1ec7n sinh v\u1eadt bi\u1ebfn \u0111\u1ed5i gen l\u00e0 r\u1ea5t quan tr\u1ecdng.<\/p>\n\n\n\n

T\u00e0i li\u1ec7u tham kh\u1ea3o<\/strong><\/h2>\n\n\n\n
    \n
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    Real-time PCR d\u1ea7n tr\u1edf th\u00e0nh m\u1ed9t ph\u01b0\u01a1ng ph\u00e1p ph\u1ed5 bi\u1ebfn v\u00e0 chu\u1ea9n x\u00e1c nh\u1ea5t trong ch\u1ea9n \u0111o\u00e1n b\u1ec7nh v\u00e0 c\u00e1c nghi\u00ean c\u1ee9u khoa h\u1ecdc li\u00ean quan \u0111\u1ebfn vi\u1ec7c khu\u1ebfch \u0111\u1ea1i v\u00e0 ph\u00e1t hi\u1ec7n t\u00e1c nh\u00e2n. B\u00e0i vi\u1ebft n\u00e0y cung c\u1ea5p t\u1ea5t c\u1ea3 th\u00f4ng tin c\u1ea7n thi\u1ebft v\u1ec1 k\u1ef9 thu\u1eadt Real-time PCR t\u1eeb th\u00e0nh ph\u1ea7n h\u00f3a ch\u1ea5t cho \u0111\u1ebfn c\u00e1ch ph\u00e2n t\u00edch k\u1ebft qu\u1ea3, … <\/p>","protected":false},"author":1,"featured_media":8063,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_uag_custom_page_level_css":"","site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"default","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","ast-disable-related-posts":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"set","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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