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Applied and Environmental Microbiology, April 2000, p. 1453-1459, Vol. 66, No. 4
Department of Food Science, Cornell
University, Ithaca, New York 14853
Received 14 June 1999/Accepted 6 January 2000
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 72 B. 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. cereus
strains 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-negative B. 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 with
B. 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.
Bacillus cereus is a
gram-positive spore-forming rod that is ubiquitous in the environment.
B. cereus has been implicated in many foodborne outbreaks
involving cooked foods such as rice, meat loaf, turkey loaf, and mashed
potatoes (9, 23). In addition to these types of foods,
B. cereus is a common contaminant in dairy products
(3). Food poisonings from the consumption of B. cereus-contaminated milk products have been rarely reported. Holmes et al. (16) reported an outbreak in which eight
people developed acute food poisoning symptoms after the consumption of
a macaroni-and-cheese dish. The epidemiological investigation resulted
in the incrimination of the macaroni and cheese based upon the
identification of high levels of B. cereus (108
to 109 organisms/g) within the food that was not served.
Bacteriological analysis of the ingredients identified the powdered
milk as the source. Schmitt et al. (20) reported two cases
of food poisoning that resulted from the consumption of powdered milk
products. B. cereus-like organisms were identified in the
milk products; however, positive identification of the isolates was not
performed (20). There is great concern, since many infant
formulas are milk based and infants have a higher susceptibility risk
than the general population (3).
Conventional methods for the identification of B. cereus
consist of biochemical tests and microscopic analysis of cell
morphology. Microscopic analysis is necessary since the closely related
Bacillus thuringiensis has similar biochemical
characteristics. Discrimination between these two species by classical
methods is based upon the visual identification of a crystalline
protein toxin produced by B. thuringiensis (24).
There are two commercially produced kits for the identification of
B. cereus, the TECRA VIA kit (International Bioproduct,
Inc., Redmond, Wash.) and the BCET-RPLA kit (Oxoid Ltd., Basingstoke,
United Kingdom). The TECRA VIA kit is specific for a 45-kDa protein of
nonhemolytic three-component enterotoxin, while the BCET-RPLA kit is
specific for the L2 protein of three-component hemolysin
(5, 14). Schraft and Griffiths (21) have
developed a PCR-hybridization assay that is specific for organisms
within the B. cereus group (B. cereus,
Bacillus mycoides, and B. thuringiensis).
We have developed a highly sensitive probe-based fluorogenic PCR assay
which can discriminate B. cereus from B. thuringiensis based upon two single-base differences within
cereolysin AB gene. Initially, the sequences of the hemolysin BL and
cereolysin AB genes were examined to differentiate B. cereus
from B. thuringiensis. These two genes were chosen based
upon their presumptive association with the ability of B. cereus to cause illness. Hemolysin BL has been shown to lyse sheep
erythrocytes and elicit a vascular permeability reaction in rabbits,
which correlates with the enterotoxigenicity of B. cereus
(12). It has been suggested that hemolysin BL is responsible
for the diarrheal food poisoning syndrome (4). The
cereolysin AB gene encodes phospholipase C and sphingomyelinase, which
constitute a biologically functional two-component cytolysin (11,
21). B. cereus phospholipase C and sphingomyelinase
act synergistically in lysing human erythrocytes (11). Three
single-base, species-specific differences between B. cereus
and B. thuringiensis were identified within the cereolysin
AB gene. A fluorogenic probe was designed based on the sequence
differences. This fluorogenic PCR assay was used to test nonfat dry
milk (NFDM) samples for the presence of B. cereus.
Bacterial strains.
The bacterial isolates used in this study
are listed in Table 1. All bacterial isolates were grown in Trypticase
soy broth (TSB) (Difco, Detroit, Mich.). A total of 72 B. cereus isolates and 5 B. thuringiensis isolates were
tested as pure cultures in this study (Table 1). Strains were
identified to species level by using a number of methods as described
in the Bacteriological Analytical Manual (1). The
putative B. mycoides strains are noted in Table 1 only on
the basis of rhizoid growth.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of a Fluorogenic Probe-Based PCR Assay
for Detection of Bacillus cereus in Nonfat Dry
Milk
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains used in the study and fluorogenic assay results
using CerTAQ-1 and CerTAQ-2
PCR for hemolysin and cereolysin sequences.
GenBank
sequences for the B. cereus hemolysin (accession no. L20441)
and cereolysin AB (accession no. M24149) genes were used to design
primers (Table 2) for the amplification
of 780- and 639-bp fragments from the hemolysin and cereolysin AB genes, respectively. All PCRs were performed in a Perkin-Elmer (Foster
City, Calif.) model 2400 thermal cycler. The PCRs were carried out in a
50-µl volume consisting of 1× PCR buffer; 1.5 mM MgCl2;
100 µM (each) dATP, dCTP, dGTP, and dTTP; 1 U of AmpliTaq DNA
polymerase (Perkin-Elmer, Norwalk, Conn.); 500 nM each primer; and 1 µl of bacterial lysate. Lysates were prepared by the
lysozyme-proteinase K treatment described previously by Czajka et al.
(6). The cycling conditions were as follows: one cycle at
94°C for 3 min; 30 cycles at 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min; and one cycle at 72°C for 10 min. PCR
products were visualized by agarose gel electrophoresis and ethidium
bromide staining.
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Cloning and sequencing of PCR fragments. PCR amplicons from B. thuringiensis strains 713 and 833 and B. cereus strains 17-B and 17-P were cloned using T vector plasmids (Promega, Madison, Wis.) according to the protocol described by the manufacturer. The sequences of the inserts were determined by dye termination using an Applied Biosystems (Foster City, Calif.) model 373 DNA sequencer and GENESCAN 672 software (Applied Biosystems).
NFDM spore counts. The number of B. cereus spores per gram of NFDM was estimated for each sample using the three-tube most-probable-number (MPN) technique as follows (24). Two grams of NFDM was resuspended in 20 ml of TSB. Ten milliliters of the suspension was removed for testing with the fluorogenic PCR assay (see below). Of the remaining 10 ml, three 1-ml aliquots were inoculated into 10 ml of TSB containing polymyxin B sulfate (100 U/ml) (Sigma) (19). The sample was then serially diluted in sterile saline. Triplicate 1-ml aliquots from each dilution tube were inoculated into 10 ml of TSB containing polymyxin B sulfate (100 U/ml) and incubated for 48 h at 30°C. Tubes showing growth were streaked on B. cereus selective agar (Oxoid) and were incubated at 30°C for 48 h. Colonies typical of B. cereus colonies (large, crenated colonies, with failure to ferment mannitol and precipitation of hydrolyzed lecithin) were microscopically examined for the presence of lipid globules, spores, and the B. thuringiensis crystalline protein toxin. The number of positive tubes for each dilution was then used to calculate the CFU per gram of NFDM according to the MPN chart.
Spiked NFDM. B. cereus isolate 17-P was inoculated into 10 ml of TSB and grown at 30°C to an optical density at 600 nm of 1.2. The culture was serially diluted, and 100-µl aliquots from each dilution were inoculated into 10 ml of reconstituted NFDM (1 g in 10 ml of TSB). One-hundred-microliter aliquots from the same dilutions were plated in triplicate on Trypticase soy agar to determine the inoculation level.
NFDM testing. One hundred microliters of trypsin (200 mg/ml) (Sigma) was added to the 10-ml sample of reconstituted NFDM set aside during the MPN preparation. The solution was then incubated at 55°C for 60 min to allow the trypsin to digest the proteins in the NFDM. The solution was incubated for 1 h at 37°C to allow for spore germination. The 1-h germination time was chosen based upon the results of In't Veld et al. (17), in which 90% of the spore population germinated within 30 min; 1 h was chosen in an attempt to achieve >90% spore germination (the 1-h incubation at 37°C was omitted for the samples spiked with vegetative cells). The solution was then centrifuged at 500 × g for 5 min at room temperature to remove any remaining particulate. The supernatant was centrifuged at 2,000 × g for 15 min at room temperature to collect the bacterial cells. The cells were washed with 1 ml of phosphate-buffered saline (pH 7.4) and centrifuged at 2,000 × g for 5 min at room temperature.
The cell pellet was then processed by the following protocol, which is a modification of the method described by Herman et al. (15). The cell pellet was resuspended in 500 µl of water, to which 100 µl of 30% ammonium hydroxide was added, and mixed by vortexing. One hundred microliters of ethanol and 3.5 µl of 10% sodium dodecyl sulfate (SDS) were added, and the solution was mixed by vortexing and then centrifuged at 12,000 × g for 15 min at room temperature. The supernatant was removed by aspiration, and the pellet was resuspended in 400 µl of a precipitation buffer consisting of 6 M urea, 1% SDS, 0.3% sodium acetate, 25% ethanol, and bacteriophage
DNA (New England Biolabs, Beverly, Mass.) (80 ng/ml). The solution was then centrifuged at 14,000 × g for 15 min at room temperature. The pellet was resuspended in 50 µl of a denaturing solution (0.1 M NaOH, 0.4% SDS) and microwave heated for eight 30-s intervals. Four hundred fifty microliters of 0.2 M NaCl was added to the microwave-treated solution, to which 600 µl
of cold ethanol was added. The solution was incubated at 
20°C for
30 min and centrifuged at 14,000 × g for 15 min at 4°C. The supernatant was removed by aspiration, and the pellet was
washed with 70% ethanol and dried under vacuum. The pellet was
resuspended in 50 µl of 10 mM Tris-1 mM EDTA (pH 8.0). Ten microliters was used for the PCR.
PCR detection with fluorogenic probe.
The probes and primer
sequences are described in Table 2. Probe synthesis (Applied
Biosystems) was described previously (2). The PCRs were
carried out in 50-µl volumes consisting of 1× PCR buffer; 4 mM
MgCl2; 100 µM (each) dATP, dGTP, and dCTP; 200 µM dUTP,
0.5 U of uracil DNA-glycosylase (New England Biolabs, Inc.), 2.5 U of
AmpliTaq DNA polymerase (Perkin-Elmer), 500 nM each primer, and 200 nM
fluorogenic probe. The cycling conditions were as follows: one cycle of
50°C for 2 min and 94°C for 3 min; 45 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min; and one hold for 10 min
at 72°C. After PCR cycling, the fluorescence intensities of the
reporter dye FAM (6-carboxy-fluorescein) (em = 518)
and quencher dye TAMRA (6-carboxytetramethyl-rhodamine)
(em = 582) were quantified with a Perkin-Elmer
LS-50B luminescence spectrometer, and 
RQ values were calculated as
previously described (2). The RQ is defined as the
difference in the ratio of the fluorescence intensity of the reporter
dye over the quencher dye for the probe in the sample as compared to
the ratio of the fluorescence intensity of the reporter dye over the
quencher dye for the probe when no target is added. A RQ threshold
was calculated based upon the 99% confidence interval above the mean
value for three no-template controls. RQ values greater than the
threshold are considered positive. The generation of the 461-bp PCR
product was verified by agarose gel electrophoresis and ethidium
bromide staining.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Hemolysin and cereolysin AB gene sequences. The hemolysin BL and cereolysin AB genes were chosen based upon their presumptive association with the ability of B. cereus to cause illness. Since both of these genes are inherently associated with the B. cereus group, we cloned and sequenced fragments of each gene to identify nucleotide differences in a collection of B. cereus and B. thuringiensis strains. A fragment of each gene was chosen so that the entire amplicon sequence could be determined by sequencing from the 5' and 3' ends of the amplicon. Sequence analysis of the 780-bp hemolysin amplicon demonstrated that there were no species-specific differences between B. cereus and B. thuringiensis (data not shown). However, three single-base, species-specific differences were identified within the 639-bp cereolysin amplicon at nucleotide locations 866, 875, and 1287 (Table 2).
Fluorogenic PCR results for pure cultures.
A total of 72 B. cereus strains and 5 non-B. cereus strains
were tested with the fluorogenic PCR as pure cultures, using the cerTAQ-1 and cerTAQ-2 probes. Table 1 lists the corresponding RQ
values and assay results. The fluorogenic PCR assay using the cerTAQ-1
probe was optimized as follows. The initial PCR conditions, consisting
of an annealing temperature of 55°C, a 1.5 mM MgCl2 concentration, and a total of 30 cycles, resulted in RQ values of
0.16 and 0.00 for B. cereus 17-P and B. thuringiensis 713, respectively. The annealing temperature,
MgCl2 concentration, and number of cycles were increased in
order to determine which conditions provided the greatest fluorescence
signal for B. cereus while maintaining a minimal
fluorescence production for B. thuringiensis. The final
reaction conditions, as described above, consisted of an annealing
temperature of 59°C, 4 mM MgCl2, and 45 cycles. A total
of 51 out of 72 B. cereus isolates were positive according to assay calculations. Of these, some B. cereus strains had
RQ values in the range of 3 to 5, while others were much lower but above the threshold value. All of the isolates used in this study were
tested with the fluorogenic PCR as pure cultures (Table 1). Forty-four
B. cereus strains were positive, with a RQ value higher than 0.4, using the cerTAQ-1 probe. Five B. thuringiensis
strains were tested, and four of the five were negative with the
cerTAQ-1 probe. One strain, B. thuringiensis 826, that
tested positive was confirmed as a B. thuringiensis strain
on the basis of crystal toxin production.
RQ value lower than 0.5 (Table 3).
Interestingly, all six isolates possessed a thymine at the 3' base
(nucleotide 876) of the probe binding site. There was also one
additional base difference at nucleotide 860 for the B. cereus 3-V sequence (Table 3). The other B. cereus
strains had sequence differences at nucleotides 859 and 860 (cytosine
and adenosine), while B. cereus strain JAP-IV was different
only at nucleotide 860 (adenine) (Table 3).
|
RQ values of 0.83 and 0.18 for
B. cereus 17-B and B. thuringiensis 639, respectively. The annealing temperature and MgCl2
concentration were varied to optimize the RQ for B. cereus while minimizing the RQ for B. thuringiensis.
The RQ values obtained with an annealing temperature of 57°C for
cerTAQ-2 are presented in Table 1. A total of 35 out of 39 B. cereus strains tested were positive, with a RQ value of >0.4,
with the cerTAQ-2 probe. The cerTAQ-2 probe could detect more B. cereus strains than the cerTAQ-1 probe, but the RQ values were
less than half of the values obtained with cerTAQ-1.
Spiked-NFDM analysis.
Reconstituted NFDM samples were
inoculated with decreasing levels of B. cereus 17-B
from an initial inoculum of 465 CFU/g. Figure
1 shows the fluorogenic PCR results for
the spiked and nonspiked samples. The average RQ values for
triplicate PCRs are shown. RQ values ranged from 2.34 to 0.61 for
samples inoculated with 465 and 58 CFU/g (the lowest inoculum level)
CFU/g, respectively. The average RQ value for the nonspiked sample
was 0.11, well below the threshold of 0.33. The RQ values showed a
linear relationship with cell numbers, indicating the possibility of
quantification of contamination levels with this assay.
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Analysis of naturally contaminated NFDM samples. Forty-three NFDM samples were tested for the presence of B. cereus by the MPN technique. Twelve of the 43 samples tested were B. cereus negative; however, two of these samples contained high levels of B. thuringiensis (93 and 1,100 CFU/g). Nineteen samples were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. The remaining 12 samples were contaminated with B. cereus at levels ranging from 43 to 460 CFU/g.
The NFDM samples were also tested with the fluorogenic PCR assay. Figure 2 shows the relationship between theRQ values from the fluorogenic PCR assay and the number of
B. cereus spores per gram of NFDM. Threshold values for the
fluorogenic PCR assay varied slightly, so the average of all of the
calculated thresholds (0.10 ± 0.03) was used in Fig. 2. All NFDM
samples that were contaminated with B. cereus at levels of 9 CFU/g or less were negative by the fluorogenic PCR assay. No samples
were found to contain B. cereus at levels between 9 and 43 CFU/g. RQ values for samples contaminated with B. cereus
at levels of 43 CFU/g (0.11 and 0.15) were just above the calculated
threshold of 0.10, suggesting a positive sample. While this level of
contamination was positive, the RQ values were lower than expected,
since the extrapolation of the sensitivity curve indicated a minimum
sensitivity of 25 CFU/g. RQ values increased proportionally to the
number of B. cereus CFU per sample to 460 CFU/g (Fig. 2).
The B. cereus-negative samples containing B. thuringiensis at levels of 93 and 1,100 CFU/g produced RQ
values of 0.00 and 0.03, respectively. These RQ values demonstrate the assay's specificity even when high levels of B. thuringiensis are present.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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B. cereus has been implicated in many food poisoning outbreaks involving rice, meat loaf, chicken, and pasta (9, 23). Food poisoning resulting from the consumption of B. cereus-contaminated milk products has not been well documented. The incrimination of B. cereus is often through epidemiological investigation, without the recovery of B. cereus from the patient, the food source, or both. While reported cases of food poisonings resulting from the consumption of B. cereus-contaminated milk products are few, the prevalence of B. cereus-contaminated milk products is quite high. Becker et al. (3) have found that 54% of dried milk products and infant formulas tested were contaminated with B. cereus at levels ranging from 0.3 to 600 CFU/g. Since B. cereus is a common contaminant in NFDM products and infant formulas are often NFDM based, regulations have been established that allow a maximum of 100 B. cereus spores/g of infant formula (10).
The detection of B. cereus in food by classical methods often requires selective enrichments of up to 48 h followed by selective plating for 24 to 48 h. Presumptive B. cereus isolates must then be tested by several biochemical and microscopic procedures to confirm whether or not the isolate is B. cereus (24). Crystal formation is one key test that positively identifies B. thuringiensis. Acrystalliferous variants of B. thuringiensis or nonrhizoid variants of B. mycoides may be misidentified as B. cereus (1). In addition to these variants, B. thuringiensis does not produce the crystalline toxin until late in the growth phase, when nutrients are diminishing. Improper growth conditions, such as a rich medium, might result in the lack of crystal production. The BCET-RPLA kit (Oxoid) and the TECRA VIA kit (International BioProduct, Inc.) are two commercially produced immunoassays that are currently available for the detection of B. cereus. Both kits require the culture of presumptive B. cereus isolates for 6 to 18 h prior to testing. The culture supernatants are then tested for the enterotoxin-related proteins. The BCET-RPLA kit uses antiserum which is specific for L2 of three-component hemolysin (containing components B, L1, and L2), while the TECRA-VIA kit uses antiserum specific for the 45-kDa component of nonhemolytic three-component enterotoxin (13, 14, 19). An evaluation of the BCET-RPLA test conducted by Granum et al. (13) resulted in the positive identification of 95% of the toxigenic B. cereus strains tested. There were two isolates associated with the diarrheal syndrome that lacked the L2 of three-component hemolysin, resulting in false-negative reactions. In addition to these two false negatives, there was one false positive resulting from an isolate that did not produce the enterotoxin, as confirmed by Western blotting, but was positive in the BCET-RPLA test (13).
Several studies have compared the BCET-RPLA and TECRA-VIA kits for their ability to detect the B. cereus enterotoxin (5, 8, 19). Buchanan and Schultz reported the positive identification of 8 out of 10 and 9 out of 10 enterotoxigenic B. cereus strains by the BCET-RPLA and TECRA-VIA kits, respectively (5). Day et al. reported that 6 of 13 enterotoxigenic B. cereus strains were positive according to the BCET-RPLA, while all 13 produced positive results in the TECRA-VIA assay (8). Rusul and Yaacob also found similar results after testing 194 B. cereus isolates. Of the 194 isolates tested, 84.5 and 91.8% were positive according to the BCET-RPLA and TECRA-VIA kits, respectively (19). All of these studies suggested that the TECRA-VIA kit has a greater ability to identify enterotoxigenic B. cereus. Aside from the study conducted by Buchanan and Schultz (5), which tested only one B. thuringiensis isolate, none of these studies tested B. thuringiensis and B. mycoides isolates to determine the rates of false-positive results produced by these species.
In this study, a highly sensitive probe-based fluorogenic PCR assay was
developed to detect B. cereus, based on species-specific nucleotides within the cereolysin AB gene. Initially, the cereolysin AB
genes of a collection of B. cereus and B. thuringiensis strains were examined, since this gene is inherently
associated with the B. cereus group. The cereolysin AB gene
encodes a two-component cytolysin, containing phospholipase C and
sphingomyelinase, which has a close relationship with Clostridium
perfringens 
-toxin (22). In addition to possessing
both cereolysin AB activities and metal binding properties, clostridial
-toxin is similar in size to the sum of mature cereolysin AB
phospholipase C and sphingomyelinase components, that is, about 43,000 Da (11, 25). With the close relationship between two
cytolysins, it was suggested that a cereolysin AB-type determinant may
have evolved to the clostridial -toxin as the result of deletion of
the intergenic spacer (766 bp) and additional sequences not required
for enzymatic activity (11). Assuming that the cereolysin AB
gene may undergo an evolutionary process, sequence divergence was
expected to occur around the intergenic spacer region of this gene.
There were no sequence differences observed within the intergenic
spacer regions of the cereolysin AB genes of B. cereus and
B. thuringiensis. Instead, two nucleotides in cereolysin A
(nucleotides 866 and 875) and one nucleotide in cereolysin B
(nucleotide 1287) were found to be specific for B. cereus.
Since these three single-base differences in cereolysin AB genes were
consistent among the tested B. cereus and B. thuringiensis strains, it is interesting to speculate that the
sequence polymorphism found in the cereolysin AB gene may reflect the
evolutionary mutagenic drift in the B. cereus group.
The ability of the fluorogenic PCR assay to specifically identify
B. cereus rather than B. thuringiensis is very
useful when testing NFDM products. As seen by the MPN testing of NFDM
samples, two samples were contaminated with high levels of B. thuringiensis, at 93 and 1,100 CFU/g. The specificity of the
fluorogenic PCR assay is a key feature, since the misidentification of
B. thuringiensis (at levels of 
100 CFU/g) as B. cereus would be a false positive.
The B. cereus fluorogenic PCR assay was able to detect at least 58 CFU/g of NFDM. Extrapolation of the sensitivity curve to the threshold (Fig. 1) results in an approximate sensitivity of 25 CFU/g. In addition to detecting as few as 25 B. cereus CFU/g of NFDM, the fluorogenic PCR assay is relatively quantitative and provides a rapid means of detecting B. cereus in NFDM compared to conventional methods that require several days of incubation or enrichment. Sample results can be obtained within 9 h from the start of the protocol. While not totally inclusive for all strains which are biochemically and microscopically identified as B. cereus, the assay did detect all of the culture-positive NFDM samples tested.
Differentiation of isolates from the B. cereus group based upon morphological characteristics is questionable at times, since there is a possibility that these characteristics may not be expressed or may even be completely lost. Isolates of B. mycoides and B. thuringiensis that lost the ability to produce rhizoid colonies and crystal toxins, respectively, have been reported (1). Without these discriminatory characteristics, the isolates would be identified as B. cereus. The difficulty in clearly identifying isolates within the B. cereus group is also increased due to the recent identification of enterotoxin-producing B. thuringiensis strains (7, 18). In light of the present findings, further research is needed to clearly determine the relationship between isolates of the B. cereus group and the possible health threats that each species might possess.
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ACKNOWLEDGMENTS |
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We thank J. Bruce and A. Miller for providing the strains used in this study and T. Arvik, N. Cady, and S. Trotter for their valuable technical assistance throughout this project.
This work was supported by the Northeast Dairy Foods Research Center.
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FOOTNOTES |
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* Corresponding author. Mailing address: 311 Stocking Hall, Cornell University, Ithaca, NY 14853. Phone: (607) 255-2896. Fax: (607) 255-8741. E-mail: cab10{at}cornell.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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