Detection of two variant vero toxin genes in Escherichia coli by capillary electrophoresis
Detection of two variant vero toxin genes in Escherichia coli by capillary electrophoresis
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DOI：
10.1002/bmc.130.abs
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发表时间：
2002-02-01
影响因子：
1.8
通讯作者：
Maeda, M
中科院分区：
文献类型：
--
作者：
Arakawa, H;Watanabe, K;Maeda, M
为了快速、特异地分析O 157的vero toxin（VT）1和VT 2基因，建立了毛细管电泳（CE）-等位基因特异性PCR、CE-单链构象多态性（SSCP）和CE-裂解酶片段长度多态性（CFLP）3种方法。等位基因特异性聚合酶链反应（PCR）方法，利用特异性双重PCR与特异性引物的VT 1和VT 2，显示VT 1和VT 2分别由174和128 bp。后续CE。进行了分析。分离时间为4 min。SSCP利用PCR方法中同时与VT 1和VT 2反应的一对引物，随后进行单链DNA二级结构的CE分析。两个基因可以在大约18分钟内分析。CFLP，像SSCP，是一种检测突变诱导的单链DNA二级结构变化的方法。核酸内切酶切割酶I识别并切割从VT 1和VT 2获得的PCR产物169 bp的自退火单链DNA中发夹环的5'侧。通过CE分析产生的DNA片段，并且电泳图显示序列特异性CFLP。分离时间为6 min。这些技术适用于O 157的检测和鉴定。版权所有（C）2001约翰威利父子有限公司
Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE. analysis was carried out. Separation time was 4 min. SSCP which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157. Copyright (C) 2001 John Wiley Sons, Ltd.