ABT® Urine Prep Solution (HI-010)

Overview 

Previously, the urinary tract was considered a sterile environment, except in cases of urinary tract infection. However, recent studies using culture and molecular biology methods have shown that urine contains a relatively small amount of bacteria (less than 10^5 CFU/mL of urine), much lower than the average of 10^12 CFU/mL found in other parts of the body such as the intestines and the vagina. Nevertheless, urine is a reliable non-invasive sample commonly used to detect sexually transmitted bacteria or parasites such as Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis through nucleic acid amplification methods. Additionally, evaluating the microbiota present in the urinary tract plays an important role in clinical screening, which is still being widely researched, such as for urethritis, urinary tract infections, and bladder cancer. Furthermore, urine collection is more acceptable to men compared to urethral swab collection. However, optimal methods for processing urine samples for PCR-based research have not been widely studied.

To address the issue of low bacterial concentration in urine, increasing urine volume (>30 mL) is an approach commonly used to enhance DNA recovery yield. However, urine typically contains high concentrations of PCR inhibitors such as urea, β HCG, and salt crystals that interfere with bacterial DNA detection. Urine is often refrigerated or frozen to prevent bacterial overgrowth and nucleic acid degradation. However, storing urine at low temperatures can lead to the precipitation of certain crystals such as calcium oxalate, uric acid, and amorphous phosphate or urate crystals, which can reduce DNA yield and inhibit molecular biology applications. Centrifuging large volumes of urine can exacerbate the problem of DNA extraction and PCR inhibition. Heating urine to dissolve these crystals is one approach, but it can also lead to bacterial cell membrane lysis, DNA degradation, or uncontrolled bacterial proliferation. Besides temperature, pH is also a factor in the formation of crystals in urine. For example, uric acid and amorphous urate crystals tend to form in acidic urine (usually pH <5.8), while calcium phosphate and amorphous phosphate crystals form in neutral or alkaline urine. Some crystals, like calcium oxalate, can form regardless of pH with sufficient concentrations of Ca²⁺ in the urine.

Products ABT ® Urine Prep Solution provides a urine processing solution containing components that dissolve salt crystals and stabilize the DNA and microorganisms present in the urine. This enhances DNA extraction efficiency and improves target detection in PCR assays compared with untreated urine.

ABT ® Urine Prep Solution

Intended use: Processing urine samples to remove salts and impurities for DNA/RNA extraction purposes.

Specification:

• The maximum volume for a processed sample: 45 mL, including: 5-40 mL urine + 5 mL ABT ® Urine Prep Solution.
• The minimum required concentration of the processing solution: 10% (v/v).
• pH of urine after processing: 6-8.
• Bacterial DNA recovery efficiency: 2-8 Ct earlier than unprocessed samples.
• Purity ratio A260/A280: 130-200% compared to unprocessed samples.
• Shelf life: 12 months from the date of manufacture
• Storage conditions: Room temperature.

Package:

Procedure

I. Processing Urine Before Storage (Method 1):

1. Collection and Processing of Urine Samples

- Collect urine into sample containers with a volume between 5-40 mL.

- Add 5 mL of processing solution to the container.

- Tighten the lid and store at a cool temperature of 2-8°C until extraction.

2. Collection of Extraction Targets in Urine

- Transfer up to 40 mL of processed urine into a centrifuge tube.

Note:

• Use a 1.5 mL tube if processing ≤1 mL of urine.
• Use a 15 mL falcon tube if processing >1 mL to 14 mL of urine.
• Use a 50 mL falcon tube if processing >14 mL to 40 mL of urine.

- Centrifuge to collect the biomass.

Note: Different centrifuge forces can be applied depending on the target for DNA extraction to achieve optimal results and suit lab conditions.

+ Bacteria, fungi: ≥ 4000×g, 10 minutes.

    + cfDNA: (refer to Supplementary Information-1).

    + Virus: (refer to Supplementary Information-2)..

- Extract according to the instructions of the extraction kits, or store at -20^oC if not immediately extracted.

1. Processing Urine After Storage (Method 2)
2. Collecting urine samples

- Collect urine into sample containers with a volume between 5-40 mL.

- Tighten the lid and store at a cool temperature of 2-8°C until extraction.

2. Urine sample processing

- Add 5 mL of processing solution to the sample container.

- Shake the sample container well and wait for 3 minutes for the crystals to dissolve.

- The urine becomes clear (or has an insignificant amount of residue). If the urine sample contains too many crystals, additional 1-5 mL of processing solution may be added.

3. Collection of Extraction Targets in Urine (Similar to Section I.2.)

NOTE:

Urine treated with ABT® Urine Prep Solution may not be suitable for biochemical tests other than DNA extraction and PCR reactions.

- Urine can be processed with a volume <5 mL, as long as the volume of added processing solution reaches 10-40% (v/v) of the final volume. For example, add 100 µL of processing solution to 900 µL of urine. However, the microbial population in urine is usually low, so too low an input volume may affect the PCR target detection capability.

- The processed urine solution may still contain a small amount of residue, dust, organic impurities, etc.

Supplementary Information:

1. For cfDNA extraction targets, the following steps can be performed:

+ Centrifuge a maximum of 5 mL/processed urine tube with ABT® Urine Prep Solution at 16000×g for 10 minutes at 4°C.

+ Carefully transfer the supernatant (4-4.5 mL) to a new tube without disturbing the pellet after centrifugation. The supernatant contains floating cfDNA.

+ Extract cfDNA from the collected supernatant according to the next instructions of the extraction kit (samples can be temporarily stored at -80°C until extraction).

2. For virus extraction targets, the following steps can be performed:

+ Centrifuge a minimum of 20 mL of processed urine with ABT® Urine Prep Solution at 4000×g for 10 minutes.

+ Then, re-dissolve the pellet after centrifugation in 5 mL using the supernatant.

+ Extract viral DNA/RNA according to the next instructions of the extraction kit (samples can be temporarily stored at -80°C until extraction).

The above procedure is only a reference for cfDNA and virus extraction; users should refer to various sources or follow the specific instructions of cfDNA and virus extraction kits.

Read more: ABT Bio-reagents ; Instructional Video for ABT Kit