Real-time PCR-based Method For The Detection Of Listeria Monocytogenes In Simulated Clinical Sample And Multi-locus Sequence Typing Of Listeria Monocytogenes

To establish a real-time PCR method for the rapid detection of Listeria monocytogenes in simulated clinical specimens.A pair of primers and one Taq-Man probe were designed based on hlyO gene.The target gene was cloned into the vector pMD18-T to make the standard curve.The specificity of the primers and probe was tested by using different serotypes L.monocytogenes strains and other common enteric pathogenic bacteria.DNA from fecal and blood samples contaminated artificially at different concentration of L.monocytogenes were extracted and tested by real-time PCR.The results of detection were positive for the samples containing L.monocytogenes,but negative for the samples with other control bacteria nucleic acid.The standard curve showed that 22 copies target genes per reaction can be detected by this method.The lowest detection limit of this method were 6.35×10~3CFU per g for fecal samples and 7.0×10~2CFU per ml for blood samples.It just took 2 hour to get the result for fecal sample and 1.5 hour for blood sample.The Taq-Man real-time PCR method for detecting L.monocytogenes in stimulated clinical specimen showed high specificity,sensitivity, simplicity and celerity.Therefore,this method can be used to identify the samples from patients infected by L.monocytogenes,and confirm the pathogen of the food borne listeriosis outbreak.A fairly high percentage of L.monocytogenes contaminated food in China.The objective of this study was to analyze the molecular epidemiologic characters of L.monocytogenes in China.A total of 183 L.monocytogenes strains were characterized by serotyping and multi-locus sequence typing.Serotype 1/2a was found in 30.1%of isolates; serotype 1/2b in 17.5%,serotype 1/2c in 31.7%,serotype 3a in 2.7%,serotype 3b in 1.1%, serotype 4b in 6%and 10%was undefined.The result of multi-locus sequence typing showed 40 different allelic combinations,the most common sequence type is ST9(26%), followed by ST8(9.3%),ST87(9.3%) and ST122(7.7%).There were 19 STs represented by only one strain.Minimum spanning tree analysis showed that clonal complex 9(CC9) was dominated by serotype 1/2c strains.There was some relationship between serotypes and sequence types because the isolates of a given clonal complex or sequence type had the same serotype.Compared to the MLST data of L.monocytogenes we found Chinese strains showed polymorphism in the molecular epidemiologic distribution.Some Chinese strains had the same sequence type with the isolates related to human listeriosis in some developed countries.As a food borne pathogen with serious public health concern,these particular sequence types L.monocytogenes strains should be attached more attention for the department of food safety and health.This study established a database about genetic diversity of L.monocytogenes isolates from food in China,which can be used in the surveillance of listeriosis.