Nongel Detection of PCR Amplicons Diagnostic of E. COLI, O157:H7: Potential for Use in Field Conditions

• Main Authors: Shaw, Joe J, Ramalka, Catherine A, Beninga, Kathy, Mcleskey, Ferne K, Hawkins, Leigh K
• Format: Report
• Language: English
• Subjects: AGAR; BACTERIA; CHAIN REACTIONS; CHROMATOGRAPHY; CLINICAL MEDICINE; DETECTION; DIAGNOSIS(MEDICINE); ELECTROPHORESIS; ENZYMES; ESCHERICHIA COLI; FIELD CONDITIONS; GELS; HYBRIDIZATION; IDENTIFICATION; Medicine and Medical Research; Microbiology; NONGEL DETECTION; PATHOGENIC MICROORGANISMS; PCR(POLYMERASE CHAIN REACTION); PLASMIDS; POLYMERIZATION; PRIMERS; SOUTH(DIRECTION); STRATEGY; VIRULENCE

The Polymerase Chain Reaction (PCR) has provided powerful new strategies for the detection and identification of bacterial pathogens. We routinely use PCR-based methods to detect, identify, and study E. coli O157:H7 in our laboratory. Generally, after PCR, agarose gel electrophoresis is performed to visualize and characterize amplicons. However, amplicons of similar sizes cannot always be differentiated by electrophoretic methods, and spurious amplicons may occasionally be generated under multiplex PCR conditions. Southern hybridization methods can be employed to clear up ambiguities, but such measures can be laborious and time-consuming. We have used a rapid paper chromatography-based method, purchased in kit form (GeneCombm, BlO 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. coil 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. coil 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.