以複合式即時聚合酶鏈反應法偵測 O157:H7 型出血性大腸桿菌

Detection of enterohemorrhagic Escherichia coli O157:H7 by using multiplex real-time PCR technology

許瀞方(ching-Fang Hsu)

指導教授 ： 潘子明

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10135

摘要

即時聚合酶鏈反應法目前被廣泛地應用在病原菌之檢驗上，本研究以複合式即時聚合酶鏈反應檢驗在培養基、糞便與食品檢體中的 O157：H7 型出血性大腸桿菌。本研究利用 2 對引子對及 2 條不同的螢光探針進行O157：H7型大腸桿菌中之目標基因偵測，包含了合成 O157 型體抗原基因 (rfbO157) 中之192 bp 片段及類志賀氏毒素第 2 型基因 (stx2) 中之 170 bp 片段。分析 217 各類菌株結果顯示，本研究根據特有的 O157 型體抗原基因以及類志賀氏毒素基因序列所設計之即時聚合酶鏈反應檢測法，能準確地檢測與區分出 O157：H7 型出血性大腸桿菌及非 O157 :H7大腸桿菌菌株，在檢測過程中，缺乏這二種基因之菌株，皆呈現陰性反應。此套即時聚合酶鏈反應方法，對於牛奶檢體不經過增殖即直接檢測時，能定量分析之濃度線性範圍為 103~109 CFU/mL，針對糞便檢體和蘋果汁檢體的定量分析之濃度線性範圍為 104~109 CFU/g (或104~109 CFU/mL)，針對碎牛肉檢體的定量分析之濃度線性範圍為 105~109 CFU/g。牛奶及蘋果汁檢體經過增殖後再檢測時，能偵測範圍可達100~104 CFU/mL。本研究中針對 O157：H7 型出血性大腸桿菌合成 O157 型體抗原基因和類志賀氏毒素第 2 型之基因，進行即時聚合酶鏈反應，不僅能快速偵測存在糞便或食品材料檢體中的 O157：H7 型出血性大腸桿
菌，同時也可進行定量分析。

關鍵字

複合式即時聚合酶食品中毒菌 ； 鏈反應法 ； O157:H7 型出血性大腸桿菌

並列摘要

Real-time polymerase chain reaction (PCR) assays have been developed for detection and quantification of pathogens in recent years. A multiplex real-time PCR assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan®) to detect enterohemorrhagic Escherichia coli O157:H7 in pure culture, feces and food samples. Two sets of primers and fluorescent probes were used for amplification and real-time detection of a 192-bp region of the rfbO157 gene encoding E. coli O157:H7-specific O-antigen, and 170-bp segment of stx2 gene encoding Shiga-like toxin 2. Analysis of 217 bacterial strains demonstrated that the multiplex real-time PCR assay successfully distinguished E. coli O157:H7 serotype from non- E. coli O157:H7 serotypes and provided accurate profiling of genes encoding O-antigen and Shiga-like toxin 2. Bacterial strains lacking these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for the two genes were linear over DNA concentrations corresponding from 103 to 109 CFU/mL of E. coli O157:H7 in pure culture and milk sample. The real-time PCR allowed construction of standard curves that facilitated quantification of E. coli O157:H7 in feces, apple juice, milk and ground beef sample. Detection sensitivity of the real time PCR assay ranged from 104 to 109 CFU/g (or 104 to 109 CFU/mL) of feces or apple juice without enrichment. Detection sensitivity of the real time PCR assay ranged from 105 to 109 CFU/g of ground beef without enrichment.After enrichment of milk and apple juice samples in modified Tryptic Soy Broth, the detection of levels were from 100 to 104 CFU/mL. The real-time PCR assay for rfbO157 and stx2 proved to be a rapid test for detection of E. coli O157:H7 in food matrices and could also be used for quantification of E. coli O157:H7 in
foods or fecal samples.

並列關鍵字

Escherichia coli O157:H7 ； multiplex real-time PCR

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