Establishment And Application Of A Method To Detect Iable Bacteria Using PMA Combinded With QPCR

PMA (propidium monoazide, PMA), a DNA-intercalating dye, can penetrate thecompromised membrane. Upon exposure to bright visible light (maximum absorbance at460nm), PMA, with the azide group, can covalent bind to DNA. With intact membranes, theviable cells can prevent PMA from penetrating the cell-wall and binding to DNA, which thedead cells and cells with compromised membranes are not able to. PMA treatment of bacterialcultures comprised of a mixture of viable and dead cells, thus leads to selective removal ofDNA from dead cells.Presented here is a method to detect live E.coli O157: H7by pretreated with PMA andsubjected to qPCR. The results indicated that, the minimum concentration to inhibit theamplification of DNA from heat-killed was10μg/mL; while the maximum concentration thathad no effect on the amplification of DNA from live bacteria was20μg/mL, and it wasenough to clear the uncombined PMA by exposed to bright light for5min. The DNA fromdead bacteria was removed from the sample by the PMA treatment when live bacteriarepresented more than1%of the total.The PMA-qPCR method was used to monitor the sterilization rate of ultra-high pressure,ultrasound, and high pulsed electric field in this study. Conclusively, the Ct value ofPMA-qPCR increased with increasing disinfection strength. The△Ct between PMA-qPCRand qPCR became larger with stronger treatment in all the experiments. The Ct value ofPMA-qPCR correlated well with lgCFU determined by plate counting. Monitoring thedisinfecting efficacy by membrane integrity seemed to be reliable.The PMA-qPCR methodwas proved applicable when used for monitoring the disinfection efficiency of the threenon-thermal processing methods. Evaluation of the influence of different factors on thesterilization rate showed that the treatment time and the power were the most importantparameters at certain temperature and ultrasonic frequency, while the duty ratio showed littleinfluence on the sterilization rate.Two typical environments were simulated:1. samples contaminated with Salmonellaenteritidis and Staphylococcus aureus;2. food samples inoculated with E.coli O157：H7. Theresult of PMA-qPCR shows no significant differences with result of plate counting. Thismethod was proved efficient when used to detect the amount of viable in these artificiallycontaminated samples.Pretreatment with PMA is easy to operate and can remove the DNA of cells withcompromised membrane effectively. PMA-qPCR method is proved capable in quantifying viable cells in complex samples.