Next Generation Direct PCR (Next Gen Direct PCR) offers unique advantages over older, less robust technologies
Next Gen Direct PCR: what is it and how does it help my studies?
Direct PCR is a means of DNA amplification directly from a test sample without performing RNA extraction or DNA extraction. As such, it is also referred to as extraction-free PCR or direct amplification.
Traditional direct PCR products often result in poor performance and cannot reproducibly process diverse samples, particularly clinical and environmental samples. Furthermore, direct PCR products using older technologies often need to be stored at -20°C, and thereby increases transportation and storage costs. These products sometimes require the user to modify clinically validated PCR test kits, resulting in clinical reference laboratories wasting time on additional validation studies. The following vendors offer direct PCR products using older technology: Promega, ThermoFisher, Zymo Research
Traditional direct PCR products often result in poor performance and cannot reproducibly process diverse samples, particularly clinical and environmental samples. Furthermore, direct PCR products using older technologies often need to be stored at -20°C, and thereby increases transportation and storage costs. These products sometimes require the user to modify clinically validated PCR test kits, resulting in clinical reference laboratories wasting time on additional validation studies. The following vendors offer direct PCR products using older technology: Promega, ThermoFisher, Zymo Research
Next Generation Direct PCR (Next Gen Direct PCR) refers to direct PCR products utilizing nanotechnology to overcome the limitations of traditional direct PCR products. In most cases, these products offer results comparable to RNA and DNA extraction.
PCRopsis™ Next Gen Direct PCR reagents allows for extraction-free PCR by utilizing proprietary nanotechnology to simultaneously perform the following tasks in a microenvironment that's compatible with PCR:
- Lyse cells & viruses
- Disrupt nucleic acid structures (tertiary and quaternary) that interfere with amplification
- Disassociate proteins bound to nucleic acids
- Stabilize nucleic acids to minimize the effects of RNases
- Sequester PCR inhibitors to ensure optimal PCR
Are all Direct PCR products the same?
Extraction-free PCR using older technologies are not able to perform all of the tasks listed above in a simple and low cost fashion. This deficit in their technological approach translates to poor performance, as observed in this report. Poor performance by traditional direct PCR products, along with other issues, has been a hinderance to wide-spread adoption of direct PCR techniques.
Next Gen Direct PCR vs Traditional Direct PCR
The table above summarized a series of quantitative PCR studies comparing PCRopsis™ Next Gen Direct PCR (Reagent RVD-RT) to Promega (XpressAmp) traditional direct PCR technology. This study used SARS-CoV-2 spiked into 11 commonly used viral transport medias and analyzed cycle threshold (Ct) values for 4 common SARS-CoV-2 gene sites (N1, N2, E and N). Viral RNA was extracted using Qiagen QIAamp Viral RNA kit as a control.
Key Points:
The full study can be viewed here.
Key Points:
- Next Gen Direct PCR results in Ct values comparable to viral RNA extraction (traditional direct PCR often results in Ct values far greater than viral RNA extraction)
- Next Gen Direct PCR results in comparable Ct values when the viral sample is in diverse transport mediums (traditional direct PCR sometimes fails to result in any amplification, leading to false negatives)
- In this study, Next Gen Direct PCR would have resulted in 100% detection of COVID-19 samples if one analyzed the N1 + N2 gene regions (commonly used in the USA)
- In contrast, Promega's XpressAmp traditional direct PCR approach would have correctly identified 6/11 samples as positive (55%). Resulting in a false negative rate of 45%!
The full study can be viewed here.
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