Development Of A Quantitative Method To Detect Viable Salmonella And Staphylococcus Aureus By PMA-qPCR In Livestock And Poultry Meat

Salmonella and Staphylococcus aureus are two most serious foodborne pathogens associatedwith human food-borne diseases. In China, food poisoning resulting from Salmonella andS.aureusis mainly due to the ingestion of contaminated livestock and poultry meat. A betterunderstanding of these two types of foodborne pathogens in livestock and poultry meatcontamination as well as obtaining theirpollution quantitative data can provide importantscientific evidence for food safety risk assessment. The aim of this study is to establish a quickand effective method to quantify viable Salmonella and S.aureus, and preliminarilyapply ittolivestock and poultry meat products to provide technical support for rapid quantitative detectionof food safety.Propidium monoazide (PMA) is a photosensitive dye that binds to nucleic acid, which canbe used for the selection of live/dead cells.By going through the incomplete membrane of deadcells, the dye can irreversibly modify DNA and prevent its amplification, while intact membraneof live cells prevents PMA from getting into the cells, thus the DNA of living cells can benormally amplified. The selective functions of PMA depend on the differences among variousspecies. In this study, two stains of Gram-negative bacteria (Salmonella) and Gram-positivebacteria (S.aureus) were choosen to analyze the influence of PMA onlive/dead cells. Several keyfactors of PMA effect were optimized, including the light exposure time, the minimum inhibitoryconcentration, and the maximum toxic concentration. It was confirmed that5minutes was theappropriate exposure time. A treatment of15μg/ml PMA can completely inhibit the DNAamplification in dead salmonella bacteria, while>40μg/ml of PMA impact the viable ones. ForS.aureus, the minimum inhibitory concentration of PMA was30μg/ml and the maximum toxicconcentration was50μg/ml.In this study, we combined PMA and qPCR to establish PMA-qPCR quantitative detectionmethod. This method not only made a better use of the rapid quantitative features of qPCR, butalso took the advantage of live/dead cell selective properties of PMA. It may circumvent theappearance of false positives and omissionin conventional qPCR methods and culture countingmethod as well as develop a rapid and accurate technology forquantitative detection of viablecells. We also assessed the reliability of the PMA-qPCR methods via testing thesensitivity andspecificity. PMA-qPCR method can detect down to101CFU/ml Salmonella or S.aureus.To establish standard curve of PMA-qPCR, we designed and builtstandard plasmid fortargeting the specific sequence of Salmonella invA gene. The cell copy number of invA gene andcycle threshold values (Ct) presented the satisfactory linear dependability and the coefficient ofcorrelation R2approached0.9979. Standard curve established by the plasmid can detect as low as10copies/reaction targeting invA gene. The plasmids containing S.aureusnuc gene specificsequence was built to establish standard curve forquantitative detection method of S.aureus and the coefficient of correlation R2approached0.9973. This method can detect as low as14copies/reaction. The construction of standard plasmid guaranteed the stability of PMA-qPCRmethod.In this study, PMA-qPCR method wasapplied to quantitatively detect viable bacteria inlivestock and poultry meat samples. The samples were artificially contaminated by Salmonella orS.aureus. The detection range was103-108CFU/mL. The difference between PMA-qPCR methodand the traditional culture counting method in salmonella was not statistically significant (P>0.05). And the difference between PMA-qPCR based quantitative detection of viable S.aureusand traditional paper counting method was also not statistically significant (P>0.05).It is a successful attempt to use PMA-qPCR for quantitative detection of viable foodbornepathogens in the meat of livestock and pourty. According to our research, PMA-qPCR technologycan effectively and quickly distinguish live and dead bacteria, and accurately quantify viablefoodborne pathogens. PMA-qPCR technology can potentiallybe used for other food-bornepathogens and has a wider prospectin the field of quantitative detection of viable foodbornepathogens.