Study On Real-time PCR For The Detection Of Listeria Monocytogenes In Meat

Listeria monocytogenes, belonging to Listeria genus, is a kind of food-borne pathogenic bacteria which can cause human and animal diseases. It is widely distributed in the environment , where its primary habitat may be soil, decaying vegetation and foods. Ingestion of foods contaminated with L. monocytogenes can result in listeriosis, a severe infectious disease characterized by meningitis, septicemia and abortion etc. The fatality rate is high (30%～40%). Listeriosis predominantly affects certain risk groups, including pregnant women, newborns, elderly people, and immunocompromised patients. The increasing incidences of L.monocytogenes in food-borne outbreaks draw people's attention. Traditional method for routine detection of L.monocytogenes is complex and time-consuming. It takes from 5 to 7 days with low sensitivity. This method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive.Real time PCR has become a routine molecular tool for detection of Food-Borne pathogens and is more expensive and time-consuming than microscopic counts, it is more sensitive, one of the major advantages of real-time PCR is that the bacterial and total microbial populations can be measured concurrently. However , We designed specific PCR primers and probes for the hlyA gene in AT-rich regions with Low signal intensity which due to poor probe detection efficiency.To overcome this problem,we improved the method of real time PCR for L. monocytogenes with Locked-Nucleic-Acid-Incorporated DNA probes. Using Ordinary method, we can not design a suitable temperature probe, by adding four LNA-based Taq Man probe integrated we can meet the design requirements of experiments both for the accuracy and reliability and the sensitivity. LNA is a bicyclic nucleic acid and can enhance oligonucleotide affinity and specificity which is a chemically modified nucleic acid with its sugar ring locked in an RNA like (C3'-endo) conformation.The introduction of LNA residues in oligonucleotides stabilizes the duplex by either preorganization or increased base stacking.In this study, the hlyA gene of L.monocytogenes was chose as target gene.,In this study, according to listeria monocytogenes(54001) strain element, specific primers and a TaqMan probe were chose. The DNA fragment of 119 bp was amplified. PCR products were confirmed by DNA sequencing. The fragment of listeria monocytogenes was cloned into T vector and transformed into JM109. The positive recombinant plasmid was used as standard quantitative template to develop a Real-time PCR.The PCR system was optimized including annealing temperature ,the ratio of the primer and probes. The reaction mixtures ( total volume, 25μL) contained the following: Premix Ex Taq12.5μL(Takara), PrimerF (10μM) 0.5μL, PrimerR (10μM)0.5μL, probe (3μM) 1μL, Template2μL, ddH2O8.5μL. The PCR protocol consistof 45 cycles of predenaturing at 95℃for 10 s, predenaturing at 95℃5 s annealing at 60℃for 30 s.The sensitivity of the Real-time PCR assay was 30 copies/μL, 1.2×10 cfu /mL; The detection limit of artificially contamination was 48 copies/μL, 3.2×102 cfu/mL .The detection could be finished in one business day.