Quantitative duplex PCR of Clostridium botulinum types A and B neurotoxin genes.
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 10(2) CFU/ mL for type A and 10(3) CFU/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 10(2) to 10(6) for type A and 10(2) to 10(7) 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.
Kasai
Yoshiaki
Y
Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology: 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan.
Kimura
Bon
B
Tajima
Yosuke
Y
Fujii
Tateo
T
D001905:Botulinum Toxins / Q000032:analysis* / Q000235:genetics; D019274:Botulinum Toxins, Type A / Q000032:analysis* / Q000235:genetics; D005504:Food Analysis / Q000379:methods; D005516:Food Microbiology; D005798:Genes, Bacterial; D009498:Neurotoxins / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D009498:Neurotoxins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A