| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 86 === B. cereus is one of the food pathogens which may cause
foodbornedisease. It is also one of the important items for food
inspection. Thus, tounderstand the relationship between its
enterotoxins and cytotoxicity andto develop the multiplex PCR
system for its detection is important. Based on the hblA
gene for B component of hemolysin BL, bceTand entFM genes for
enterotoxins, PCR primers aimed for the detectionof B. cereus
enterotoxins have been developed. When the PCR resultsfor these
enterotoxins specific primers were compared with the results
ofcytotoxicity study using CHO cells, it was found that strains
are positivewith any of these PCR primer pairs would show the
positive cytotoxicityresult. The results for CHO cells
cytotoxicity study were the same asthose obtained from
immunoassay studies using BDE-VIA kit. As forthe multiplex PCR
system, it was found that for hemolysin BL or bceTgene
detection, when only one target strain was present in skim milk
andcooked rice, the detection sensitivity reached to N*100 CFU
per ml andper gram if a preculture step was performed prior to
PCR. When twotarget strains were mixed to the food sample,
however, it was found thatthe ratio of original cell numbers
should be within 102 so that the targetstrains with lower cell
numbers could be detectable. On the work forspecific PCR
detection of B. cereus (not the B. cereus group cells),
afterseries designing of PCR primers from 16S rRNA genes, we
found PCRprimers which could allow the specific detection of B.
cereus butexempted the interference from B. anthracis, B.
mycoides and a standardstrain of B. thuringiensis. Four B.
thuringiensis strains which interferethe PCR detection of B.
cereus strains, however, were confirmed as B.thuringiensis
strains since they generate cry gene specific product andthey
were shown to produce parasporal crystals as examined by
themicroscope. Sequence assay for the 16S rRNA genes showed that
thesefour B. thuringiensis strains have the same DNA sequcnce as
B. cereusstrains. Furthermore, on the position of base No. 165,
two bases, ie. Cand T were observed. Whether two or more 16S
rRNA operons would befound for B. thuringiensis strains need to
be further investigated.
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