Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50820
標題: Enteroaggregative E. coli in vitro adherence test, hemagglutinating test etc, development and use of PCR primers for the detection of EAggEC
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
作者: Tsai, Cheng-Chih
蔡政志
關鍵字: Enteroaggregative E. coli
腸凝集性大腸桿菌
PCR
cell culture
bacteria clump formation test
hemagglutination test
multiplex PCR
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
出版社: 食品科學系
摘要: 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.
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。
URI: http://hdl.handle.net/11455/50820
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