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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.

Air-Dryable Direct DNA qPCR Urine, MDX150

Available in 5 mL (1,000 Rxns) or 50 mL (10,000 Rxns) aliquots

Air-Dryable Direct RNA/DNA qPCR Urine, MDX151

Available in 5 mL (1,000 Rxns) or 50 mL (10,000 Rxns) aliquots

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.

冻干后的预混液在正确封存的情况下可在常温下稳定保持2年。

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.

常见问答: 开发风干分子检测的重要基本原理

风干是通过蒸发过程移除反应液体中的水分。该过程需要使用精密的烘箱, 通过优化的干燥温度和时间来实现充分干燥。不同的反应预混液有不同的理想失水率,当失水率达到这个水平,表明干燥完成。 在相应的产品操作指南中我们推荐了对应的烘箱温度和干燥时间。但值得指出的是,许多因素会影响风干过程,如反应容器、反应液体积、风干批次的大小和预混液的最终配方(例如,是否添加了引物探针和添加了多少体积的引物探针),因此需要根据具体情况进行风干优化。

可以的, 我们建议根据不同的反应配方(比如说风干反应液是否含有引物探针,以及添加引物探针的体积)、使用的反应容器(孔板或试管)、风干批次大小等因素,优化烘箱温度和干燥时间。高温有可能会影响反应液及其组分的完整性,包括酶的活性。

理想情况下,风干MDX095 可风干一步法RT-qPCR预混液应移除70%的水分(仅对预混液进行风干时, 风干MDX082可风干qPCR预混液应移除95%的水分(仅对预混液进行风干时)。因此,每个检测反应需要优化风干设置来达到这个数值。如果水分残留过多可能会影响风干试剂的保质期,而过度干燥可能会使风干混合物很难复溶,因此导致性能下降。 失水率可以通过对比风干前后含有预混液的反应管重量来计算 (请参阅基本流程进一步了解相关信息)。此外,风干后必须要立即封装风干混合物。

风干试剂在添加样本后可在秒内迅速复溶。 建议加入样本后轻晃或震荡反应管使反应液充分混匀。

如果反应液被过度干燥,会导致酶的性能受到影响。理想的风干状态应该是达到建议的失水率,酶的性能依然不受影响。 如果风干试剂的Ct值延迟,这通常表明试剂被过度风干,干燥过程需要优化。

我们没有发现使用不同荧光探针 (Cy5, FAM, JOE 或ROX) 对风干试剂的性能产生影响。

我们在风干过程中没有观察到任何污染,但是强烈建议遵守良好的实验室清洁规范以减少可能的环境污染。例如,保持操作表面清洁、将做风干干燥、qPCR反应配置和数据分析的区域分开。此外,建议遵照制造商提供的说明维护清洁烘干箱。在干燥过程中,风扇产生的空气流动是否会将少量引物/探针从一个

我们没有观察到任何由空气流动造成风干过程中的交叉污染。风干混合物在试管底部,反应管之间发生交叉污染的可能性极小。此外,出于GMP规范原则,不建议将不同测试的反应液在同一烘干过程中同时进行干燥。

也可以使用其他反应容器,如板孔、小瓶或各种微芯片,但不同反应容器或检测的干燥条件需要优化。确定最佳干燥条件和参数后,需要进行验证,包括扩大生产规模。

我们建议使用精密烘箱(温度均匀性+/- 1.5°C),可以有鼓风或真空的功能。 在我们的测试中,使用了Memmerd UF260 Plus烤箱,风扇和风门的设置分别为100%和20%。请注意,不同的烘箱有不同的规格和可调参数,需要根据具体检测、风干液体积、反应容器类型、风干批次大小、相对湿度和海拔高度进行优化。

如果首次风干失败,没有达到正确的失水率,我们不建议把试管再放回烘箱继续风干或者加水延长风干。为了保证风干参数可控性和检测的可重复性,建议重新配置风干。除此之外,在风干完成前不建议在风干过程中打开烘箱门,因为会影响烘箱中温度等环境。

风干与冻干不同,因此失水率不会受环境影响。

当体系中引入任何变量时,如反应体积、反应容器、引物/探针或批次大小,我们建议分开测试。这将确保条件的可控性和结果的可复制性。

不。天平不需要靠近烤箱,只要盖上管盖,然后用天平测量即可,即使天平摆在另一个房间也可以。  

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