Molecular Typing And Invasiveness Genes Research Of Listeria Monocytogenes

In accordance with the national standard (GB/T4789.30-2008), from June2011to June2012in Shanghai,479samples were collected from supermarkets and vegetable market which includedpoultry, meat, dairy products,34Listeria monocytogenes were isolated (7.1%).34isolates weredivided into four categories by multiplex PCR serotype, serotype1/2a&3a accounted for76.47%,serotype1/2c&3c accounted for17.65%, serotype1/2b,3b&7accounted for2.94%, the serotype4b,4d&4e accounted for2.94%.1/2a&3a is weak pathogenic serotypes which accounted thelargest (76.47%). While the the strong pathogenicity serotype4b,4d&4e only accounted2.94%.The biofilm forming ability of34isolates were measured by a96-well microplate assay. Biofilmformation capacity experiment showed that100%of isolates were capable of forming biofilm, whereas the strong biofilm is not formed by strong pathogenic4b, but by weak pathogenic1/2a&3a type.MLST method divided34isolates into eight types: ST121, ST155, ST11, ST8, ST196, ST9, ST87, ST589. LM19did not get the appropriate type. Remaining33isolates, ST121accounted for45.5%, followed by the ST155(15.2%), ST11(12.1%), ST8(9.1%), ST196(6.1%), ST(6.1%), ST87(3.0%), ST589(3.0%). According to comparative genomics analysis between Listeriamonocytogenes and innocua. Two suspected invasion and adhesion related genes lm1290and lm2027were selected to invest. According to the genome of10403s, primers of lm1290, lm2027weredesigned, SOE-PCR amplification of homologous recombination arm, and cloned into E.coli-listeria temperature sensitive shuttle plasmid pKSV7, get a recombination plasmid pK1290-B1-B2; transformed into10403s, after antibiotic resistance and temperature sensitive screening, alm1290deletion mutant was confirm by PCR testing. A10403s transformed with pK2027-B1-B2,but the filter is not successful. Identification of wild strains and lm1290deletion mutant strain growthcurve, RT-PCR was used to detect the expression of regulated gene prfA. And through cellexperiments and biofilm assessment, to prove the invasion change between the wild-type strain anddeletion mutant.