A Novel, Rapid, and Simple PMA-qPCR Method for Detection and Counting of Viable Organisms.

INTRODUCTION

The plate counting method widely used at present to discern viable from non-viable in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable organisms.

MATERIAL AND METHODS

Using propidium monoazide (PMA) to inhibit amplification of DNA from dead , a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable was established. The standard recombinant plasmid with the target gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed.

RESULTS

The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R) of the standard curve was 0.999. The sensitivity of the method was 10 CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude.

CONCLUSION

In this study, a rapid and simple PMA-qPCR counting method for viable was established, which will facilitate related research.