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研究生:謝尤敏
論文名稱:台灣地區引起食品中毒之仙人掌桿菌其腸毒素圖譜、PCR檢測與分子分型
論文名稱(外文):Enterotoxigenic profiles, PCR detection and molecular typing for foodpathogenic bacillus cereus strains in Taiwan
指導教授:曾浩洋曾浩洋引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:仙人掌桿菌腸毒素細胞毒性分子分型法
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摘要
仙人掌桿菌(Bacillus cereus)是台灣地區重要食品病源菌之一,此菌在營養生長時產生的腸毒素會造成腹瀉型食品中毒症。在仙人掌桿菌群中除仙人掌桿菌外,還有蘇力菌(B. thuringiensis)、蕈狀桿菌(B. mycoides)與炭疽桿菌(B. anthracis)。為了解仙人掌桿菌群菌株的致病能力,設計不同引子組進行四種腸毒素-Hemolysin BL(HBL), Non-hemolytic enterotoxin(Nhe), Enterotoxin FM(EntFM)及 B. cereus enterotoxin T(BceT)基因的PCR擴增。2株炭疽桿菌無任何一種毒素基因之擴增產物,89株仙人掌桿菌,7株蘇力菌及3株蕈狀桿菌皆能擴增出至少一種腸毒素基因,其中30株仙人掌桿菌及所測試的7株蘇力菌、3株蕈狀桿菌具有hbl操縱子中的三組基因-hblA、hblC及hblD,仙人掌桿菌菌株BCN32能檢測出hblC及hblD但無hblA基因,除仙人掌桿菌菌株BCN32與BCN58有hbl基因外,但BCET-RPLA套組的檢測卻為負反應,其餘hbl基因PCR檢測結果與使用BCET-RPLA套組的檢測結果大致相同。BceT基因在101株仙人掌桿菌群中有45株檢出,entFM基因則有95株菌株能擴增出目標產物,nhe操縱子的檢出率(96%;97/101)最高,以四種腸毒素的檢測結果,可將101株仙人掌桿菌群菌株分成八種腸毒素型,其中最常見的是Profile III,同時具有entFM及nhe基因,有41株仙人掌桿菌(41﹪)。分析仙人掌桿菌群菌株上清濾液對CHO、HEp-2及Vero三細胞株的細胞毒性,仙人掌桿菌、蘇力菌與蕈狀桿菌菌株皆有細胞毒性,炭疽桿菌則無細胞毒性,三細胞株中以HEp-2細胞對於仙人掌桿菌的毒素敏感性最低。仙人掌桿菌菌數需達>5 × 107 cfu/ml以上才能檢測出細胞毒性,細菌培養至穩定期時細胞毒性會下降21 ~ 23。為了解腸毒素基因hbl及nhe的多形性,以PCR-RFLP分析及部份基因序列對,發現兩基因皆有變異存在,但基因序列仍具有相當保留性,多數鹼基對取代是發生在密碼子的第三個位置,並不影響胺基酸序列的表現。根據hbl操縱子中三基因多條序列比對取得親緣關係樹狀圖,可見仙人掌桿菌與蘇力菌的親緣關係很近,而仙人掌桿菌BC13則與蕈狀桿菌菌株BMY1與BMY2則顯示為同一群,應歸於蕈狀桿菌。經由抗生素敏感性分析,質體圖譜,隨機擴增形性分析(RAPD)與脈衝式膠體電場電泳分析83株仙人掌桿菌之分子類。抗生素敏感性分析可得24種抗生素敏感性圖譜,質體圖譜有46型,其中27株仙人掌桿菌仍分離出質體。以NotI剪切之PFGE圖譜有58型,另以5組不同引子組進行RAPD分析,則可得到42至55種不同圖譜,在仙人掌桿菌菌株BCN29 ~ BCN58之區分上,RAPD與PFGE圖譜分析結果大致符合,RAPD具有快速及簡單的優點,而PFGE則是區分性與再現性最高的分類方式。
Abstract
Bacillus cereus is one of the major foodborne pathogens in Taiwan. The diarrheal type of diseases is attributed to enterotoxins produced during vegetative growth of B. cereus. The B. cereus group comprises B. cereus, B. thuringiensis, B. mycoides and B. anthracis. The virulence properties of B. cereus group cells are of interest. One hundred and one strains of B. cereus group were examined for the presence of four enterotoxin genes: the hemolysin BL (hbl), the non-hemolytic enterotoxin (nhe), the Bacillus cereus enterotoxin T (bceT) and enterotoxin FM (entFM) using polymerase chain reaction (PCR). Different PCR primers were developed for the detection of these genes. Two B. anthracis strains were found to be PCR-negative for these four enterotoxin genes. At least one of the four enterotoxin genes was detected in any of the 89 B. cereus, 7 B. thuringiensis and 3 B. mycoides strains. Thirty of 89 B. cereus strains, all of the 7 B. thuringiensis and 3 B. mycoides strains carried hbl operon genes i.e., hblA, hblC and hblD. Two genes of hblC and hblD were detected in B. cereus strain BCN32, but hblA was not found. The results from the amplification of hblC correlated well with results obtained with the BCET-RPLA kit (Oxoid; Denka Seiken, Japan). Except for two strains (B. cereus strains BCN32 and BCN58), all strains that were positive in PCR amplification using primers L2F/L2b were also positive when tested with the BCET-RPLA kit. The bceT gene was found in 45 of the 101 strains of B. cereus group and entFM in 95 strains. The nhe operon was the most common enterotoxin gene detected in strains examined (97/101; 96%). These results showed that there were 8 enterotoxigenic profiles for the 101 strains of B. cereus group collected. Profile III that carrying the entFM and nhe genes was the most prevalent type (41/101; 41%). Culture supernatants from all strains of B. cereus, B. thuringiensis and B. mycoides were cytotoxic to HEp-2, CHO and Vero cells. Of the three cell lines tested, the HEp-2 cell was less susceptible to the enterotoxin of B. cereus than the CHO and Vero cells. Cell cytotoxicity was detectable only after the cell concentration of B. cereus reached >5 × 107 cfu/ml and the Cytotoxicity was about 1/2 ~ 1/8 at the stationary phase. PCR-RFLP analysis and PCR-directed sequencing were performed to examine the heterogeneity of the hbl and nhe genes. The PCR products of hbl genes were found to be heterogeneous by the PCR-RFLP analysis (17/40; 42%). PCR-RFLP analysis also confirmed that there was genetic diversity within the nheA and nheB genes. The sequences different genes of hbl and nhe were found to be highly conserved for different strains. However, most of the base pair substitution occurred at the third base position of the genetic codon and did not affect the amino acid sequence. The hblA, hblC and hblD sequences were used to construct a phylogenetic tree. The strains of B. thuringiensis and B. cereus were closely related species, also B. cereus strain BC13 and B. mycoides strains BMY1, BMY3 were revealed to be closely related species. B. cereus strains were typed by antibiotic susceptibility testing, plasmid profile, RAPD (Randomly Amplified Polymorphic DNA) and PFGE (Pulsed-field gel electrophoresis).Of the 83 B. cereus strains tested, there were 24 antibiogram and 46 plasmid profiles. Plasmid was not founded in 27 B. cereus strains. Digestion with NotI generated 58 PFGE profiles for these 83 B. cereus strains. RAPD with 5 different primers yielded 42 to 55 RAPD patterns. Results obtained from RAPD and PFGE were closely related among the B. cereus strains BCN29 ~ BCN58. Although the RAPD method was rapid and easier to perform, PFGE produced most discriminative and reproducible results.
目錄
摘要
Abstract
第一章.文獻整理
一.桿菌屬(Bacillus spp)的分類
二.仙人掌桿菌(B.cereus)的一般特性
三Bacillus cereus group
四.B.cereus 引起之食品中毒症與其他臨床症狀
五.Bcereus的毒性因數
(一)嘔吐型毒素(Emetic toxin)
(二)腹瀉型腸毒素(Diarrheal enterotoxin)
(三)其他毒素分子
1.溶備素(Hemolysis)
2.磷脂?(phospholipases C)
六.B.cereus毒素檢測方法
(一)嘔吐型毒素 
(二)腸毒素
七.脈動式膠體電泳(Pulsed-field gel electrophoresis.PFGE)
八.隨機擴增多形性DNA(Randomly Amplified Polymerohic DNA:RAPD)
九.質體圖譜的分析(Plasmid profilc analysis)
十.抗生素敏感性分析(Antibiotic susceptibility testing)
第二章仙人掌桿菌腸毒素之檢測
壹.前言
貳材料與方法
一.實驗材料
1.菌株
2.培養基
3.動物細胞株
4.溶液之配置
5.儀器
6.藥品
二.實驗方法
1.DNA之抽取
2.PCR引子組
3.聚合?連鎖反應
4.不連續溶血試驗
5.BCET-RPLA套組之檢測
6.細胞毒性分析
參.結果與討論
一.仙人掌桿菌HBL腸毒素的檢測
二.不連續溶血試驗
三.仙人掌桿菌其他腸毒素之基因檢測
四.仙人掌桿菌之細胞毒性分析
肆.結論
第三章.HBL腸毒素與Nhe腸毒素基因之PCR-restriction Fagment length polymorphism分析及部分序列分析
壹.前言
貳材料與方法
一.實驗材料
1.菌株
2.DNA與PCR產物回收與純化套組
3.藥品
4.儀器
二實驗方法
1.PCR產物的純化
2.限制?剪切PCR產物
3.PCR產物定序
4.定序分析
參.結果與討論
一.HRI腸毒素基因
1.PCR-RFLP之分析
2.PCR產物之序列分析
3.PCR-RFLP與基因序列之比對
4.Hm1/Hm2引子組PCR產物之序列分析
二.Nhe腸毒素基因PCR-RFLP分析與序列分析
肆.結論
第四章.仙人掌桿菌分子分型之研究
壹.前言
貳材料與方法
一.實驗材料
1. 菌株
2. 緩衝溶液之分配
3. 藥品
4. 儀器
二.實驗方法
1.抗生素敏感性分析
2.脈動式電場膠體電泳圖譜
3.隨機擴增多形性DNA(PAPD)分析法
4.質體抽取
參.結果與討論
一.抗生素敏感性分析
二.質體圖譜分析
三.PAPD圖譜分析
四.PFGE圖譜分析
五.抗生素敏感性圖譜.質體圖譜.PAPD圖譜與PFGE圖譜的比較
肆.結論
參考文獻
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