Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk

A fluorogenic probe-based PCR assay was developed and evaluated for its uti lity in detecting Bacillus cereus in nonfat dry milk. Regions of the hemoly sin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three s ingle-base differences in two B, cereus strains were identified in the cere olysin AB gene at nucleotides 866, 875, and 1287, while there were no speci es-consistent differences found in the hemolysin gene. A fluorogenic probe- based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Ta g polymerase, and two fluorogenic probes were evaluated. One fluorogenic pr obe (cerTAQ-1) was designed to be specific for the nucleotide differences a t bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus s trains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B, th uringiensis strains tested positive. Sequence analysis of the negative B. c ereus strains revealed additional polymorphism found in the cereolysin prob e target. A second probe (cerTAQ-2),vas designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cel eus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1, A PCR assay using cerT AQ-1 was able to detect approximately 58 B, cereus CFU in 1 g of artificial ly contaminated nonfat dry milk. Forty-three nonfat dry milk samples were t ested for the presence of B. cepeus with the most-probable number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of t hese samples tested positive with the fluorogenic PCR assay. Of the remaini ng 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were ne gative in the fluorogenic PCR assay. Although not totally inclusive, the PC R-based assay with cerTAQ-1 is able to specifically detect B, cereus in non fat dry milk.