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研究生:蔡政志
研究生(外文):Tsai, Cheng-Chih
論文名稱:腸凝集性大腸桿菌之吸附試驗,血凝集性試驗等及其特異性PCR引子組之設計與應用
論文名稱(外文):Enteroaggregative E. coli in vitro adherence test, hemagglutinating test etc, development and use of PCR primers for the detection of EAggEC
指導教授:曾浩洋曾浩洋引用關係
指導教授(外文):Hau-Yang Tsen
學位類別:碩士
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:1998
畢業學年度:86
語文別:中文
論文頁數:153
中文關鍵詞:腸凝集性大腸桿菌聚合酉每鏈反應組織培養細菌團形成試驗血凝集試驗多套式聚合酉每鏈反應
外文關鍵詞:Enteroaggregative E. coliPCRcell culturebacteria clump formation testhemagglutination testmultiplex PCR
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腸凝集性大腸桿菌是造成嬰兒下痢的原因之一。目前已知腸凝集性大腸桿
菌在組織細胞上呈現之凝集附著性為其重要特徵,近年來的研究顯示,腸
凝集性大腸桿菌具有一種特殊 "凝集附著纖毛",為造成其以似疊磚方式
凝集附著於細胞表面的主要原因,且此纖毛構造基因在腸凝集性大腸桿菌
株彼此具相當高之同質性。本研究目的即在針對已發表的aggR基因以及
aggA基因序列設計出具專一性的PCR引子組,區分以Schmidt et al.設計
pCVD432引子所篩選出之7株疑似EAggEC菌株;並與組織培養之傳統檢測、
細菌團形成試驗、血凝集試驗與DNA的定序作比較,了解其PCR引子組
pCVD432、AggR-1/ R-2、AggRI/ RII、AggR5/ R3和AggA1/ A2的正確性,
並期望能與EPEC之PCR引子組合併,建立短時間檢測EAggEC/ EPEC之多套
式PCR系統。另外,應用針對腸毒性大腸桿菌毒素基因之LTI1/ I2和
STII1/ II2兩對引子組之多套式PCR反應同時檢測飲用水及環境水中之腸
毒性大腸桿菌。經由基因序列比對,本實驗室自行設計具檢測專一性的
AggRI/ RII與AggA1/ A2 引子,實驗用的7株疑似EAggEC菌株只有4株產生
大小為610和352 bp之片段。另設計一組AggR5/ R3引子,則7株菌皆產
生355 bp之預期產物,其結果與Schmidt et al.設計pCVD432引子和
Tsukamoto設計之AggR-1/ R-2引子相同。經組織培養檢測、細菌團形成試
驗及血凝集試驗確認,發現AggRI/ RII與AggA1/ A2 引子之正確性高於
AggR5/ R3引子。且相同PCR條件下,其他的菌株包括腸毒性大腸桿菌、腸
侵入性大腸桿菌、腸病原性大腸桿菌及其他非致病性大腸桿菌及非大腸桿
菌之腸內菌等以此為PCR引子均無PCR反應產物。AggA1/ AggA2 引子針對
目標菌之檢測靈敏度可達100 ~ 101之生菌數,應用於牛奶、水源與嬰兒
糞便中腸凝集性大腸桿菌檢測,若配合以MacConkey broth預培養8 ~ 9小
時,PCR檢測靈敏度可達100之原生菌數。合併AggA1/ A2和BFP1/ 2引子所
發展之多套式PCR,在純菌系統中的EAggEC和EPEC檢測上,其檢測靈敏度
可達103/ 103之等菌量生菌數。最後,以LTI1/ I2和STII1/ II2兩對引子
組之多套式PCR反應檢測台中市飲用水以及台中縣(市)附近環境水,經增
菌培養8小時,ETEC菌株T02和T10之檢測靈敏度都可達到100之原菌數。
Enteroaggregative E. coli(EAggEC) is a common cause of infantile
diarrhoea. In vitro study, shows that EAggEC strains established
characteristic "stacked-brick" on the surface of tissue culture
cells and such adhrence pattern was termed as aggregative
adherence(AA). Recently, Natatro et al. and Savarino et al.
found that EAggEC expressed an inducible aggregative adherence
fimbriae I(AAF/I) associated with the presence of AA on HEp-2 or
HeLa cells. The cloned regulator gene aggR and structural gene
aggA encoding AAF/I were found to be specific for EAggEC and not
in other enteropathogens. The purpose of this study is to design
PCR primers for the specific detection of the aggR and aggA
genes of 7 EAggEC strains which were screened by the pCVD432
primers which were designed by Schmidt et al.. The results
obtained were compared with the in vitro HeLa cells adherence
test, bacteria clump formation test, hemagglutination test and
DNA sequencing of suspected agg genes so that the accuracy of
primers pCVD432, AggR-1/R-2, AggRI/RII, AggR5/R3, and AggA1/A2
were checked. In addition, the multiplex PCR systems for the
simutaneous detection of EAggEC and enteropathogenic E. coli(
EPEC) cells were also developed. On the other aspect, combined
LTI and STII gene specific PCR primers were combined into a
multiplex PCR system and used for the simultaneous detection of
LTI and STII ETEC cells contaminated in tap water and
environmental waters. By comparison of the sequences of aggR and
aggA genes from GenBank/EMBL, primers AggRI/RII and AggA1/A2
were designed for the specific detection of aggR and aggA genes
of EAggEC. Only four strains of seven EAggEC-like strains showed
the 610 bp and 352 bp PCR products positive results with AggRI/
RII and AggA1/A2 as PCR primers. On the other hand, all of the
seven EAggEC-like strains produced expected 355 bp PCR products
with AggR5/R3 primer sets. Similar positive PCR results were
obtained when reporeted pCVD432 and AggR-1/R-2 primers were
used. Based on the above facts, in vitro HeLa cells adherence
test, bacteria clump formation test and hemagglutination test
were thus used to check the seven EAggEC-like strains. Results
showed that AggRI/RII and AggA1/A2 primer pairs developed by us
are more specific than pCVD432, AggA1/A2 and AggR-1/R-2 primer
pairs. Under the PCR conditions as described, bacteria strains
including enterohemorrhagic E. coli(EHEC), enterotoxigenic E.
coli(ETEC), enteroinvasive E. coli(EIEC), EPEC, non-pathogenic
E. coli and non-E. coli strains including strains of the family
of enterobacteriaceae, would not produce the specific PCR
product. The detection sensitivity of AggA1/A2 primers(50 pmole
each) was 100-101 CFU. In detection of EAggEC cells in milk
samples, tap water, ground water and infantile stool samples,
the detection sensitivity would be 100 CFU if a 8~9 hr pre-
enrichment step using MacConkey broth was performed prior to the
PCR. The specific PCR system for EAggEC detection could be
combined with BFP1/2 primers a multiplex PCR system. The
detection sensitivity for EAggEC and EPEC cells with the
multiplex PCR system using AggA1/A2 and BFP1/2 primers was 103/
103 for each target cells per assay. Finally, LTI1/I2 and STII1/
II2 primers were combined into a multiplex PCR system and used
for the inspection of tap water and environmental waters near
Taichung City. On detection of ETEC T02 and T10 cells in tap
water and environmental waters, if a 8 hr pre-enrichment step
was performedprior to PCR, the detection sensitivity for target
cells in water would be reach to 100 CFU per assay.
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