Detection and identification of pathogenic escherichia coli by using multiplex polymerase chain reaction and analysis of ITS(internal transcribed spacer)nucleotide sequences

• Main Author: 李偉基
• Other Authors: 張甘楠
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/89845605087225624464

碩士 === 國立屏東科技大學 === 獸醫學系 === 91 === The final goal of this study is for the fast diagnosis of different types of pathogenic Escherichria coli including EHEC ( Enterohemorragic E.coli )、ETEC ( Enterotoxigenic E.coli )、EIEC ( Enteroinvasive E.coli )、 EPEC ( Enteropathogenic E.coli ) and EAEC ( Enteroaggregative E.coli ). Moreover , the target genes employed for the differentiation of these kind of pathogenic E. coli are VTⅠ, VTⅡ, ST, LT, ial , pCVD432, eaeAgen , H7 and uidA. However, the resulting data could provide with that the pathogenic E. coli can be diagnosed by using three tubes-one step multiplex PCR ( polymerase chain reaction ). The diagnostic process just took as short as two and half hours in terms of fast- diagnosis. The ITS ( Internal transcribed spacer ) region of DNA fragment was cloned and sequenced from EHEC, ETEC, EPEC, EIEC and EAEC respectively. After a phylogenic analysis by using DNA star softwire, three groups of distinct characteristics of E. coli were demonstrated as groupⅠ: ETEC and EHEC were characterized by secreting their specific enterotoxins or cytotoxins in clinics; group Ⅱ: EAEC alone was not found in humans other than animals; group Ⅲ: EIEC and EPEC: the pathogenesis of this group of E. coli was not proposed by the mechanism of toxin-producing. The resulting data showed that 10% divergence in DNA sequences between groupⅠand groupⅡ, 41.5% divergence between groupⅠand groupⅢand the same result as shown between groupⅡand groupⅢ. However, The sequences of ITS could be used for differentiation of different types of pathogenic E. coli based on phylogenic analysis and also coincided with the characteristics of the different pathogenic E. coli which are grouped described above.