Design and validation of a multiplex polymerase chain reaction for the identification of enterotoxigenic Escherichia coli and associated colonization factor antigens

Development of a genetic tool for the detection of genes encoding enterotoxins and colonization factors would greatly enhance enterotoxigenic Escherichia coli (ETEC) surveillance. Oligonucleotide primers were designed to amplify genes encoding human ST, porcine ST, LT and the structural genes of colonization factor antigen (CFA)/I, CS1 to CS8, CS12 to CS15, CS17 to CS22, and PCFO71. Screening 89 ETEC isolates phenotypically expressing a known CFA showed that, without exception, the multiplex polymerase chain reaction (mPCR) detected the structural gene of the expressed CFA, in addition to CS21 in 22.5% of isolates. Silent genes such as cssB (CS6) were also detected in 9.0%. Additionally, we screened 71 CFA phenotypically negative isolates and detected a CFA in more than 50% of tested isolates. In conclusion, we have designed a simple 4-step mPCR for the rapid detection of ETEC virulence factors. The assay is rapid, reproducible, relatively inexpensive, and has the potential to be field applicable.