Multiplex Pcr Detection And Molecular Typing About Shigella Spp

Shigellae is the pathogen that induces bacillary dysentery which was confirmed the second serious infection disease by the Chinese government in 1985.With the improvement of living standards and strengthening of management,its incidence rate is declining,but still much higher than developed countries.Shigellae's pathogenicity and drug resistance is relative with its virulence genes.There are many researches about virulence genes of Shigellae and their detection is also one of the routine monitoring,but most of them concentrated on a single PCR,time-consuming and laborious.A variety of molecular biology typing methods,but non of them is standard.In view of this,this experiment is to develop a method of multiplex PCR to detect virulence genes(set1B,sen,ipaH,ial,stx1 and virA) in Shigella spp and compare several typing methods in order to find which is more suitable for molecular detection of Shigellae.MethodsIn order to establish the multiplex PCR,116 shigella isolates were recovered from Shigella monitor sets of Henan province during 2001～2007(1 strain S.dysenteriae,114 strains S.flexneri,11 strains S.sonnei).These bacteria are also typed by repetitive extragenic palindromic PCR(REP-PCR),random amplified polymorphic DNA(RAPD),plasmid profile and pulsed-field gel electrophoresis(PFGE) techniques and analyzed with BioNumerics software.ResultsEither 6 virulence genes were detected by multiplex PCR at the same time or detected separately by general PCR and it showed that both ways were sensitive and specific.The positive rates of sen,ipaH,stx1,ial,virA and set1B were 88.70%,99.13%,0,88.70%, 92.17%and 85.22%respectively,and the number of their type was 10.REP-PCR's positive rate was 96.57%and 0～10 bands,which's molecular weight was between 100bp and 2000bp,were amplified.116 shigella isolates were typed 3 kinds by REP-PCR.RAPD typed well,but it was very unstable.Plasmid profile typed well too,but its repeatability was not very high.All the isolates display 86 different X-balI-PFGE patterns and their repeatability was high.ConclusionsMultiplex PCR should be applied for suspicious shigella clinical samples and for the cases of outbreak and aggregation,PFGE should be applied.