Establishment Of Duplex Real-Time Pcr For Detection Of Listeria Monocytogenes And Staphylococcus Aureus In Meat

Foodborne illnesses associated with the two foodborne pathogens of Listeria monocytogenes and Staphylococcus aureus occur at high frequencies, which can result in severe influence upon human health. L.monocytogenes can cause foodborne listeriosis, such as septicemia, encephalitis, meningitis, abcess, pregnant women obortion and so on. Its mortality rate is high up to30%to70%. It can still grow and reproduct in the foods which are stored in the refrigerator at4℃further increasing the dangers. S.aureus is the most common one of causing foodborne staphylococcal diseases and can cause vomiting, diarrhea and other symptoms. L. monocytogenes and S. aureus exist widely in the environment and a range of sources, e.g. meat, milk, eggs, aquatic products and vegetables are constantly subjected to the pollution of these two bacteria to different degrees, of which meat and meat products are contaminated most seriously. Therefore, the establishment of the fast and reliable method of detecting these two pathogens in meat has. an important practical significance. The real-time PCR technique integrates the efficient amplification of PCR, the high specificity of nucleic acid probe hybridization and high accuracy of the quantitation of the spectroscopic techniques. The multi-channel excitation light sources of the quantitative PCR instrument can achieve the purpose of the simultaneous detection of a variety of bacteria. So far, the simultaneous detection of L. monocytogenes and S. aureus in meat by the real-time PCR technique has not been reported. Therefore, in order to be able to detect fastly and accurately these two bacteria in meat so as to achieve the purpose of prevention and control of diseases caused by these two bacteria, the dulplex real-time PCR for detection of L. monocytogenes and S. aureus in meat was studied.we established the real-time PCR assays for the detetion of L. monocytogenes and S. aureus respectively by selecting the hlyA (L. monocytogenes) and nuc (S. aureus) genes as the target genes and designing a pair of primers and probe according to hlyA gene and a pair of primers and probe according to nuc gene. The real-time PCR assays were developed on the basis of building positive recombinant plasmids respectively, and then the specificity,sensitivity and repeatability were tested. The results of the specificity test for detecting L. monocytogenes by real-time PCR showed that all of the strains of L. monocytogenes (n=10) were positive and all other nonspecific strains (n=6) were negative and the results of the specificity test for detecting S. aureus by real-time PCR showed that all of the strains of S. aureus (n=5) were positive and all other nonspecific strains (n=6) were negative. The results of the sensitivity tests for detection of L. monocytogenes and S. aureus indicated that the correlation coefficient of the standard curves were0.998(R2=0.998) and0.997(R2=0.997), respectively and the sensitivities were39copies and37.4copies, respectively. The results of repeatability tests of the real-time PCR methods we had established for detection of L. monocytogenes and S. aureus respectively showed that the coefficients of variation within the groups were both less than1%and the coefficients of variation among the groups were both less than2%.The duplex real-time PCR assay was developed for simultaneous and quantitative detection of L. monocytogenes and S. aureus in a single tube on the basis of the single real-time PCR assays. And then the cross-reactivity between them was analyzed and the sensitivities were detected by achieving the standard curves of the duplex real-time PCR method. The results showed that the respective amplification efficiency of L. monocytogenes and S. aureus was independent in the established duplex real-time PCR method. The correlation coefficients of the two standard curves were both above·0.99. The sensitivities of the duplex real-time PCR method for detecting L. monocytogenes and S. aureus were19.5copies per μL and18.7copies/μL respectively.Furhter, the duplex real-time PCR method for detection of L. monocytogenes and S. aureus in meat was established by applying the established duplex real-time PCR assay to detect L. monocytogenes and S. aureus in artificially inoculated meat samples and the minimum detection limit of the two pathogens in the simulative meat samples was determined. The results indicated that a range linear relationship of the duplex real-time PCR method for detection L.monocytogenes and S. aureus in meat from106cfu/mL to101cfu/mL based on plate counts, respectively, was observed between threshold cycle (Ct) and logarithmic concentration of the serial dilutions and the detection limits were both10cfu/mL. The whole testing process of the established approach took about three hours, including the extraction of the genome DNA. The approach was highly specific, sensitive and rapid, which could be applied to simultaneous and quantitative detection of L.monocytogenes and S. aureus in meat.A total of100fresh meats obtained from the market in Harbin were detected by the established duplex real-time PCR method for detection of L. monocytogenes and S. aureus in meat and the traditional culture method. The results showed that the detection rates of the duplex real-time PCR method were7%for L.monocytogenes and6%for S. aureus, being higher than that3%for L. monocytogenes and3%for S. aureus of the traditional culture method.