Quantitative duplex PCR of Clostridium botulinum types A and B neurotoxin genes

Abstract

A duplex quantitative polymerase chain reaction (PCR) assay for Clostridium botulinum types A and B was developed. The sensitivity and specificity of the assay were verified by using 6 strains of type A, 7 strains of type B, and 14 genera of 42 non-C. botulinum types A and B strains, including C. botulinum types C, D, E, F, and G. In pure culture, the detection limit was 102 CFU/mL for type A and 103CFU/mL for type B. In mushroom broth, increases in the amounts of C. botulinum types A and B could be monitored separately (the quantifiable range was 102 to 106 for type A and 102 to 107 for type B) from each sample that contained a large number of background bacteria, and toxin could be detected much earlier than with mouse assay. These results suggest that duplex quantitative PCR methods are useful to detect and quantify C. botulinum types A and/or B toxin genes.