Toxigenicity, partial 16S rRNA Sequence Comparison and the Use of a Multiplex PCR System for Bacillus cereus Group Cells

• Main Authors: Chen, Yan-Lian, 陳炎鍊
• Other Authors: Hau-Yang Tsen
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/42593639360374484716

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.