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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

Air-Dryable Direct DNA and RNA/DNA qPCR Urine graph
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.

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

FAQs: qPCR Urine Test

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.

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.

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.

We have used up to 25% human urine with our mix.

It is stable for up to 2 years at ambient temperature if correctly stored in sealed pouches.

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.

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.

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

No, air-drying requires specific stabilizers, excipients, and preservatives which are different from those required for lyophilization.

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.

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.

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.

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.

We did not observe any effect of the fluorophore type (Cy5, FAM, JOE or ROX) on the performance of the mix after air-drying.

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.

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.

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.

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.

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.

Unlike the effects with lyophilization, there is no impact of the environment to the moisture loss with air-drying.

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.

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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