A propidium monoazide-polymerase spiral reaction (PMA-PSR) designed for direct detection of Escherichia coli O157:H7 viable cell

Abstract

Escherichia coli O157:H7 is a major foodborne pathogen causing food poisoning. Standard detection method for foodborne E. coli is plate counting, which is time consuming and omits VBNC cells. PSR is an isothermal amplification technique identifying trace amount DNA. However, its effectiveness is influenced by residual DNA from dead cells. This study aimed at designing a PMA-PSR system for accurate determination of viable cells of E. coli O157:H7. Firstly, PSR was optimized in fluorescence indicator, betaine concentration and reaction time. The optimized system could successfully identify rfbE, stx1 and stx2 genes within 45 min at 65 °C. Secondly, sensitivity and specificity were tested in pure culture and different food samples. With 100% specificity, PSR could determine DNA amount as low as 1.12 pg/μL. Thirdly, a PMA-PSR system was designed for accurate determination of viable cells. Cells were treated with 5 μg/mL PMA to remove residual DNA from dead cells. Pretreated cells were adapted to PSR, thus viable cells were specifically detected. Fourthly, the system was applied in food samples with VBNC cells. Viable cells could be accurately determined in accordance with regular assay. In conclusion, the PMA-PSR system was an efficient tool for accurate determination of E. coli O157:H7 viable cells.