John Czajka

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

ABSTRACT A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereusstrains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negativeB. cereus 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 cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated withB. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 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 negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

Development of a solid-phase fluorescence immunoassay for the detection of Salmonella in raw ground turkey

A solid-phase fluorescence sandwich immunoassay was developed to detect Salmonella spp. and tested for sensitivity on pure cultures as well as on spiked and naturally contaminated raw ground turkey. The solid support was a soft-glass capillary tube to which a polyclonal antibody against Salmonella spp. was adsorbed. The adsorbed antibody captured cellular antigens from the sample solution. The same polyclonal antibody was also biotinylated and, in combination with an avidin-Cy5 dye conjugate, served to allow detection of the immunologically captured cells. The minimum detectable number of Salmonella cells spiked into ground turkey was approximately 2.5 CFU/25 g. Fifty-one raw ground turkey samples were tested for the presence of Salmonella spp. using both this immunoassay and a modified Bacteriological Analytical Methods enrichment method. Fluorescence intensity values were normalized as a ratio of the sample fluorescence to the fluorescence of a standard. Fluorescence intensity ratios of 0.50 or greater were scored as Salmonella positive. The solid-phase fluorescence immunoassay yielded 10% false positives and no false negatives when results were compared with those from the modified enrichment method. Isolates recovered from the samples that produced false-positive results were identified as Citrobacter freundii and Enterobacter cloacae , both of which are known to cross-react with the antiserum used.

Ligase-Mediated Detection Techniques

Since its introduction in 1985, the polymerase chain reaction (PCR) has revolutionized molecular biology and has facilitated the development of a variety of nucleic acid-based detection systems for genetic disorders as well as for bacterial, viral and other pathogens. Ligase chain reaction (LCR) is a recently developed DNA amplification technique, which utilizes a thermostable ligase and allows the discrimination of DNA sequences differing in only a single basepair. A measure of the intellectual genesis of LCR can be traced back to the oligonucleotide ligation assay (OLA) (Landegren et al 1988; Nickerson et al 1990). This method was used in conjunction with a primary PCR step to screen for sickle cell anemia and other human hereditary disorders. Compared to the OLA, the LCR provides a much higher degree of sensitivity and is less susceptible to false-positive ligation product formation. The use of a thermostable ligase minimizes target independent ligation since the ligation reaction can be performed at or near the melting temperature (TM) of the oligonucleotide primers (Barany 1991a).