Air-Dryable™ Direct DNA and RNA/DNA qPCR Stool
Air-Dryable™ Direct DNA qPCR and RNA/DNA qPCR Stool mixes are glycerol-free and contain optimized excipients compatible with air and oven drying. The mixes have been designed for the direct detection of DNA and RNA from stool specimens without extraction. Can also be used wet and/or with purified nucleic acid
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Air-Dryable Direct DNA qPCR Stool, 4x, MDX140
Inhibitor tolerance to 20% stool, 2 mg/mL bile salt and 40% Cary Blair media. Perfect for multiplexing and fast cycling conditions. Used in the detection of IBD, cancer, fecal microbiota and bacterial and viruses.
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Air-Dryable Direct RNA/DNA qPCR Stool, 4x, MDX141
Inhibitor tolerance to 5% stool, 2 mg/mL bile salt and 40% Cary Blair media. Perfect for multiplexing and fast cycling conditions. Used in the detection of IBD, cancer, fecal microbiota and bacterial and viruses.
Documents & Resources
Description
PCR inhibitors found in stool specimens, such as bile salts, polysaccharides, hematin, and catabolic substances, have posed challenges to developing assays that can directly amplify DNA or RNA. In addition, due to the high complexity and heterogeneity of fecal matter, sample preparation has traditionally been required to remove possible interfering substances such as food debris, microorganisms, desquamated epithelial cells, and mucus from the specimen. Air-Dryable Direct DNA qPCR and RNA/DNA qPCR Stool mixes are designed for the direct qPCR/RT-qPCR analysis from stool samples, requiring minimal sample processing. The mix contains an optimized blend of additives to negate inhibitor effects while maintaining the quality and integrity of the patient sample. As the need for fast, non-invasive testing for gastrointestinal conditions increases, short turn-around times, higher sensitivity and a long shelf-life become important distinguishing features.
If you are developing an infectious disease assay for parasites, viruses, bacteria, or a screening assay for inflammatory bowel disease or gastric or colon cancer, Air-Dryable Direct DNA qPCR and RNA/DNA qPCR Stool mixes will speed up your assay development time. The mix only requires the addition of primers and probes to create a liquid or ambient-temperature stable assay that can be dried in a laboratory oven, avoiding the need for expensive lyophilization.
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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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