RT-qPCR Protocol Optimization Assessment with Propidium Monoazide for Viable and Non-Viable Pathogen Detection

Abstract

Real time PCR or RT-qPCR (including reverse transcriptase PCR) is a gold standard in diagnostic testing for pathogens such as bacteria and viruses that contain RNA or DNA for amplification. As a validated molecular assay technique, RT- qPCR has been instrumental in microbial detection because of its high throughput, quantification of DNA or RNA samples as well as quick turnaround especially when compared with culturing methods that can take multiple days to generate result. While the advancement of PCR has had significant impact for diagnosis of diseases such as COVID- 19through its high sensitivity and specificity, the test also brings with it its own challenges of false positive and false negative results. This is normally attributed to PCR's susceptibility to contamination or inhibitors. However, there are other conditions like DNA extraction techniques and protocols in PCR methodology that can impact the accuracy of the PCR results as well. This paper, therefore, aims to synthesize factors that contribute to false positive and negatives in PCR results, improvements to protocols that includes the use of DNA intercalating dyes such as Propidium Monoazide (PMA) in PCR assay to improve DNA extraction and discuss the importance of an optimized PCR assay for diagnostic testing efficacy from a social and economic standpoint.