Nongel Detection of PCR Amplicons Diagnostic of E. Coli, O157:H7: Potential forUse in Field Conditions

摘要

The Polymerase Chain Reaction (PCR) has provided powerful new strategies for thedetection and identification of bacterial pathogens. We routinely use PCR-based methods to detect, identify, and study E. coli O157:H7 in our laboratory. We have used a rapid paper chromatography-based method, purchased in kit form (GeneCombm, BIO RAD), to rapidly and easily analyze PCR-generated products. The method is based upon chromogenic detection of amplicons generated through the use of biotinylated primers, coupled with a brief (30 min.) chromatography step. This method allows complete clinical analysis of PCR amplicons in as little as one hour, providing positive identification of E. coli 0157:H7. Multiple amplicons can be definitively detected depending upon the primers employed, the PCR conditions, and design of the chromatography step. We have used the method to easily, rapidly, and positively identity the 90-100 kb virulence plasmid of E. coli 0157:H7, and to successfully differentiate between two similar-sized amplicons of the Shigella-like toxins (SLT I and SLT II) of 0l57:H7 strains.