Abstract:
Objective To develop a method of multiplex PCR to detect E.coli O157:H7 rapidly, specifically and sensitively.To compare its sensitivity with conv entional PCR(only one sets of primers).
Methods Four pairs of primers were designed from O antigen, H flagellar antigen and Shiga-like T oxin(SLT)1 and 2 genes.Fourty strains of O157:H7 and non O157:H7 were detected by conventional PCR and multiplex PCR amplification.And the sensitivities of PCR were estimated when the str ain was diluted from 10 1-10 6.
Results O antigen and H flagellar antigen genes amplification generated amplicons of both 497bp and 625bp in all EHEC O157:H7 strains, SLT 1 and(or)SLT 2 genes amplification generated amplicons of 210bp and(or)484bp in toxinogenic O157:H7.Other non O157:H7 failed to yield any amplicon under comparable conditions.The sensitivity of detection by conventional PCR was shown to be at least 150CFU per PCR tube and > 1 500CFU by multiplex PCR.
Conclusion This multiplex PCR method was more specifically, rapidly, efficient after enrichment than traditional bacterial detection.It would become a new method to diagnose the toxinogenic O157:H7 and nontoxinogenic strain.
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