Development of a DNA-Probe for Detection of Enterotoxic Bacillus cereus Isolated from Foods in ThailandC. Wiwat* and R. Boonchaisuk
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Polymerase chain reaction (PCR) and DNA probe assays were compared for detection of B. cereus isolated from foods. The entFM gene encoding the enterotoxin FM was chosen as the target gene. Two pairs of primers, TY123/TY125 and TY123/TY127, were used to amplify DNA fragments of 584 bp and 1,219 bp, respectively. The 1,219 bp PCR product was amplified from 41 (42%) of 97 B. cereus isolates and 5 B. thuringiensis isolates. The 584 bp PCR product of B. cereus ATCC 14579 was cloned and labeled with digoxigenin (DIG)-dUTP to be used as a DNA probe. Colony blots, dot blots and Southern blots gave the same DNA hybridization results. Ninety-five isolates (98%) were positive including 56 isolates that had previously given negative PCR results. Two exceptions were B. cereus G157/44 and B. cereus 11929. Additionally, 5 isolates of B. thuringiensis gave positive results. All 3 blotting methods gave negative hybridization results for other Bacillus species and non-Bacillus species tested. The probe was able to detect 62.5 ng of B. cereus genomic DNA.
Keyword:
B. cereus, enterotoxin gene, detection, PCR, DNA hybridization, Enterotoxic, Food
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