臺灣博碩士論文加值系統

仙人掌桿菌是引起食品中毒的主要病原菌之一，亦為食品檢測之重
要病原菌，所以了解仙人掌桿菌群及其腸毒素和細胞毒性的關係，並發展
其多套式PCR的檢測方法有其重要性。 根據hemolysin BL之B
component hblA，腸毒素bceT及entFM基因，設計其腸毒素基因之PCR引子
。經過三組腸毒素基因引子的PCR檢測結果與CHO cells毒性分析的結果比
較，發現B. cereus只要其中一腸毒素基因之PCR結果為正反應，則此菌株
亦會對CHO cells產生細胞毒性。CHO cells毒性試驗結果與Bacillus
Diarrhoeal Enterotoxin Visual Immunoassay (BDE-VIA)套組檢測結果
相符合。在多套式PCR應用於食品檢測的結果發現，不管是產生hemolysin
BL 或 腸毒素bceT 的仙人掌桿菌，其單一菌株感染脫脂鮮乳和米飯並經
預培養後，多套式PCR 的檢測靈敏度均可達到N*100每毫升或每公克之B.
cereus原菌數。當二株不同的產毒性仙人掌桿菌混合時，其菌量相差102
倍時，低菌量的菌株就檢測不到其腸毒素基因的PCR產物。將B. cereus從
B. cereus group中區分亦是本研究另一個目的，經過數組針對16S rRNA
基因所設計的引子，其PCR結果發現B. cereus可以排除B. anthracis，B.
mycoides 和標準菌B. thuringiensis的干擾，而4株非標準菌B.
thuringiensis菌株，經過B. thuringiensis之cry基因引子之PCR和結晶
毒蛋白的顯微鏡觀結果，確認菌株無誤；然後，其特異性DNA探針和DNA定
序的檢測結果，得知其16S rNRA之PCR引子的基因序列有變異且基因序列
與B. cereus相同，另外此4株B. thuringiensis菌株其16S rRNA基因
之165處，其PCR產物的DNA定序結果有二個band (C和T) 是否具有不同的
operon仍須進一步探討與研究。

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.