Effect of Propidium Monoazide in the Detection of Enterotoxigenic Escherichia coli in the Pediatric

Abstract

Objectives: The purpose of this study is to determine the viability of enterotoxigenic Escherichia coli (ETEC) in a sample of diarrhea. The investigation focuses specifically on the lt gene and utilizes propidium monoazide (PMA) and quantitative real-time polymerase chain reaction (qPCR) to differentiate between live and dead bacteria. Methods: Propidium monoazide is a chemical that can bind to and inhibit the amplification of free DNA during qPCR analysis. In this study, in addition to analyzing diarrhea samples, artificially spiked samples were used to assess the sensitivity and accuracy of the PMA treatment. The qPCR results were compared to the gold standard of culture-based methods both with and without PMA treatment. Results: The method’s limit of detection was 8 CFU/mL, and it exhibited linearity from a 10-1 to a 10-9 dilution. The qPCR approach revealed a higher bacterial count than the culture method due to the detection of DNA released from dead bacteria. However, when PMA was employed, the bacterial count was similar to that obtained using colony count agar, which is attributed to the elimination of free DNA during investigation. Conclusions: The present study developed a PMA-based qPCR approach that enables the detection of live bacterial DNA. This method involves PMA and real-time PCR and offers several advantages, including faster detection times (a few hours vs. several days with the traditional culture method) and the ability to exclusively detect live bacteria without interference from free DNA released by dead bacteria. Additionally, the use of real-time PCR enables precise quantification of the live bacterial load. Overall, this approach is cost-effective, rapid, highly sensitive, and specific, making it a valuable tool for various applications.