Air-Dryable™ Direct DNA and RNA/DNA qPCR Urine Master Mixes
Air-Dryable™ Direct DNA qPCR and RNA/DNA qPCR Urine mixes are glycerol-free mixes that contain optimized excipients compatible with air and oven drying. The mixes have been designed for the development of quantitative PCR urine test assays for direct detection of DNA and RNA from human urine specimens without extraction. Can also be used wet and/or with purified nucleic acid
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Meridian's Air-Dryable™ Direct DNA and RNA/DNA qPCR Urine Master Mixes
- Create your ultra-sensitive multiplex direct quantitative PCR urine test using crude urine samples.
- Simplify qPCR urine testing, by removing the nucleic acid extraction step or inhibitor carryover during purification, shortening sample-to-result and reproducibility.
- Designed for the direct detection of bacteria and cell-free nucleic acids at very low titers.
- Can be used wet or it can be dried, for developing an ambient-temperate stable assay.
High performance (sensitivity and reproducibility) in the presence of 5% human urine
Activity of air-dried Air-Dryable™ Direct RNA/DNA qPCR Urine (red) was compared to Reliance 1-Step Multiplex RT-qPCR Supermix (purple) for their ability to amplify a 1,000-fold dilution of an RNA target (10,000 and 10 copies respectively) in the presence of 5% human urine. The results illustrate that the dried mixes maintain the same high performance as the liquid mixes and shows much higher sensitivity than Reliance 1-Step Multiplex RT-qPCR Supermix.
Air-Dryable Direct DNA qPCR Urine, 4x, MDX150
Inhibitor tolerance to 60% urine. Perfect for multiplexing and fast cycling conditions. Used in the detection of urinary track infections, STD, tropical diseases, cancer screening, bladder and prostate cancer.
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Air-Dryable Direct RNA/DNA qPCR Urine, 4x, MDX151
Inhibitor tolerance to 20% urine. Perfect for multiplexing and fast cycling conditions. Used in the detection of urinary track infections, STD, tropical diseases, cancer screening, bladder and prostate cancer.
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Description
Urine is an ideal clinical specimen because it is excreted in large quantities is non-invasive, and it can be self-sampled. Currently, urine specimens are used in the diagnosis and management of infectious diseases (including STDs), hormone and metabolic disorders, renal diseases, bladder cancer, urinary tract infections (UTIs), and for monitoring recreational drug use. However, urine contains substances such as urea and nucleases that can damage DNA or inhibit the PCR reaction.
Meridian’s new Air-Dryable™ Direct Urine molecular mixes are unique in that they enable the highly sensitive, very fast detection of target DNA or RNA directly from high concentrations of urine and are sensitive enough to detect arboviruses, such as Chikungunya virus. These mixes have been formulated specifically to overcome the inhibitors found in urine – no further optimization is required aside from the addition of primers and probes. Furthermore, these mixes can be used in a wet format or dried down by oven or air drying to create ambient-temperature stable assays.
Specifications
| Description | Glycerol-free qPCR and RT-qPCR mix containing Taq polymerase, reverse transcriptase, reaction buffer, dNTPs, MgCl2 and air-dry compatible excipients, developed to tolerate the effects of the inhibitors present in urine samples. |
| Concentration | 4x |
| Appearance | Clear, colorless solution |
| Hot Start | Antibody mediated |
| Application | Probe-based, real-time PCR, two-step RT-qPCR, one-step RT-qPCR |
| Sample type | cDNA, crude or purified RNA and/or DNA from human stool |
| Presentation | 1 vial |
| Storage | -20 °C |
| Mix stability | See outer label |
| Assay stability | Up to 24 months at ambient temperature following air-drying. |
| Consistency | ± 0.5 Ct variance between test and reference sample |
| DNase/RNase Contamination | No detectable degradation |
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FAQs: qPCR Urine Test
What are the advantages of using urine over other sample types?
The advantages of a quantitative PCR urine test are that it is completely non-invasive, and it can be self-sampled in large quantities. Urine testing is used in the diagnosis and management of infectious diseases (including STDs), hormone and metabolic disorders, renal diseases, bladder cancer, urinary tract infections (UTIs) and for monitoring recreational drug use.
What types of infectious diseases can urine detect?
The most common infections are urinary tract infections (UTIs), most commonly Escherichia coli, Protus mirabilis, Enterococcus faecalis, Staphylococcus saprophyticus and Klebsiella pneumoniae. Several other infections can be diagnosed by urine DNA and urine RNA tests including community-acquired pneumonia (CAP), legionellosis, tuberculosis, congenital cytomegalovirus (CMV) infection, and arboviruses, such as Chikungunya virus, dengue virus and Zika virus. Parasites can also be diagnosed from urine testing for the detection of eggs from Schistosoma haematobium. Furthermore, urine has been used for screening of different sexually transmitted diseases (STD) such as Neisseria gonorrhoeae and Chlamydia trachomatis.
What type of cancer can be detected in urine?
Cancer cells or their DNA or RNA are too big to pass through the kidneys and will have to come from the bladder or ureters, so bladder cancer is the most obvious urine cancer, however prostate and cervical cancer are also detectable.
What is the maximum volume of urine you can use in a quantitative PCR urine test?
We have used up to 25% human urine with our mix.
After drying for how long will the mix stay stable to use? Or do we need to use it immediately after drying?
It is stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.
Is a pre-treatment needed if you want to detect bacteria in urine?
That will depend on the bacteria, for some bacteria you may not need to do any pre-treatment, however some may require heating to 95°C for 5 min and for some tougher bacteria, a Proteinase K step (Proteinase K 55°C for 15 min and then at 98°C for 5 min) or using a non-ionic detergent (such as Tween-20 to final concentration of 0.2% followed by incubation at room temperature for 10 min) may be necessary.
What is the performance difference for DNA when using the DNA-specific urine mix versus the DNA/RNA urine mix?
We do not see a performance difference between the two mixes, which is why we call the Air-Dryable Direct RT-qPCR Urine mix – Air-Dryable™ Direct RNA/DNA qPCR Urine mix, as it can be used for both.
Are reaction conditions the same for air-dried and liquid mixes?
Yes, the Air-Dryable™ Direct DNA qPCR Urine and Air-Dryable™ Direct RNA/DNA qPCR Urine can be used as a liquid mix or air/oven dried and stored for up to 24 months without needing to change the reaction conditions and air drying/ambient temperature storage will not affect the sensitivity of the test.
FAQs: Important Principles for Air-Drying Molecular Assays
Can I use the Lyo-Ready mix for air-drying?
No, air-drying requires specific stabilizers, excipients, and preservatives which are different from those required for lyophilization.
Is it possible to adjust the oven temperature and do high temperatures damage the polymerase or reverse-transcriptase?
Yes, we recommend optimizing the oven temperature and drying time for each new assay formulation (mix including primers and probes), vessel used and lot size. High drying temperatures may affect the integrity of the mix and its components, including enzymes.
How do you know once drying is complete?
Ideally, 70% moisture should be removed from Air-Dryable™ 1-step RT-qPCR Mix (MDX095) and 95% from the Air-Dryable™ Mix (MDX082). However, the optimal moisture loss requires optimization for each assay. Retaining too much moisture may impact the shelf-life of the assay and over-drying may make the mixture difficult to rehydrate and cause a loss of performance. Moisture loss can be calculated by comparing the weight of the initial wet mix in the vessel to the dry mix (refer to the basic workflow for further information). Please note that the air-dried material must be packaged immediately after the drying cycle.
Is it easy to rehydrate an air-dried mix? Do you need to vortex?
The air-dried mix rehydrates in seconds after the addition of the sample. We do recommend, if possible, to gently shake/mix the vessels in order to resuspend the reaction mix before running the reaction, or alternatively mix the solution when adding the patient sample.
Why do I sometimes see a lower Ct value when I compare the performance of the re-hydrated dry mix against the wet mix?
If a mix becomes over-dried, the integrity of the enzymes will be compromised. Conceptually, the ideal “dryness” of a mix will be the highest percentage of moisture loss achieved without losing assay performance. If the Ct value of your dry mix is lower to the comparable wet mix, then the mix has been over-dried and further optimization is required.
Do different probes affect the performance of the assay after the mix is air-dried?
We did not observe any effect of the fluorophore type (Cy5, FAM, JOE or ROX) on the performance of the mix after air-drying.
Will the air circulation from the fan in the oven cause contamination?
We did not observe any contamination during air-drying however we do recommend good laboratory cleaning practices to minimize possible environmental contamination. Examples include surface cleaning or allocating separate locations for the oven drying, qPCR reaction setup, and analysis. In addition, we recommend maintaining the oven following the cleaning instructions provided by the manufacturer.
Could the fan air bring traces of primers/probes from one tube during the drying to another tube?
We did not observe any cross-contamination from fan-forced oven drying. Given the mix resides at the bottom of the vessel, it is very unlikely that cross-contamination can occur between the wells. In addition, in accordance with good manufacturing practices, we would discourage oven drying two different types of assay in the same drying cycle.
Do I have to use 8-well PCR tube strips?
Other vessels can be used such as plates, vials or various microchips, but the drying conditions for each vessel/assay would need to be optimized. Once the ideal conditions and parameters are established, they would need to be verified during validation and scale up.
Do you have a list of ovens that you recommend?
We recommend using a precision drying oven (temperature uniformity of +/- 1.5°C) with convection, fan-forced or vacuum capabilities. In our testing, we used a Memmert UF260Plus oven with fan and air flap settings at 100% and 20% respectively. Please note that different ovens will have different specifications and adjustable parameters that will need to be optimized based your assay, reaction volume, vessel type, batch size, relative humidity, and altitude.
Can I simply put the tubes back in the oven for longer incubation if the moisture loss is not enough, or add water if it is too much?
If the first attempt is not successful and does not provide the correct moisture loss value, we do not recommend putting the tubes back in the oven for additional drying or adding water. We recommend re-starting the experiment in order to control all of the variables so that they are reproducible. In addition, opening the door during the drying process is also not recommended unless it is done in a controllable manner and will be part of the process moving forward.
Would fluctuations in ambient humidity and temperature where the drying oven is located affect the drying protocol, such that the moisture lost is outside the 70% range?
Unlike the effects with lyophilization, there is no impact of the environment to the moisture loss with air-drying.
Can I test using different vessel types and/or different reaction volumes per well in the same drying cycle?
We recommend that different drying cycles are performed to test the effect of altering any assay variable such as vessel type, reaction volume, primer/probes, and lot size. This will ensure that the conditions and outcomes are reproducible.
Does the balance need to be next to the oven for quick weighing?
No, the balance does not have to be next to the oven, just close the vessel once it is out of the oven and go to the balance (even if in a different room) for measurement.
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