Detection of Listeria monocytogenes by Real-time Fluorescent PCR

[Objective]To develop a rapid and accurate real-time fluorescent PCR method for detection of Listeria monocytogenes.[Method]By using hlyA gene of L.monocytogenes as target sequence to design primers and adding four LNA-based TaqMan probe to the primers,a new real-time fluorescent PCR assay was developed and applied to detecting the positive plasmids and the simulated specimens.[Result]The hlyA gene was amplified from genomic DNA of Listeria monocytogenes and cloned into pUC119 to generate positive plasmid pUC-hlyA,which was used to develop the new method,the standard curve was Y=-3.273 X +37.640,R2=1.000.The sensitivity of this method was 1.2×10 CFU/ml;the detection limit of artificially contamination was 3.2×102 CFU/ml.[Conclusion]A real-time fluorescent PCR assay for the detection of Listeria monocytogenes was developed,which supplied a technology platform for the rapid detection of L.monocytogenes.