Brandal Lin Thorstensen, Lindstedt Bjørn-Arne, Aas Lena, Stavnes Trine-Lise, Lassen Jørgen, Kapperud Georg
Division of Infectious Disease Control, Norwegian Institute of Public Health, Geitmyrsveien 75, P.O. Box 4404 Nydalen, Torshov, N-0403 Oslo, Norway.
J Microbiol Methods. 2007 Feb;68(2):331-41. doi: 10.1016/j.mimet.2006.09.013. Epub 2006 Oct 31.
A multiplex PCR assay, amplifying seven specific virulence genes and one internal control gene in a single reaction, was developed to identify the five main pathotypes of diarrheagenic Escherichia coli and Shigella spp. The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC). Each forward primer was labelled with a fluorochrome and the PCR products were separated by multicolour capillary electrophoresis on an ABI PRISM310 Genetic Analyzer (Applied Biosystems). If present, several gene variants of each virulence gene were identified. The internal control gene rrs, encoding 16S rRNA, was amplified in all 110 clinical strains analyzed. Virulence genes were demonstrated in 103 (94%) of these strains. In the majority of the cases (98/103, 95%), classification obtained by the novel multiplex PCR assay was in agreement with that previously determined by phenotypic assays combined with other molecular genetic approaches. Numerous multiplex PCR assays have been published, but only a few of them detect all five E. coli pathotypes within a single reaction, and none of them has used multicolour capillary electrophoresis to separate the PCR products. The octaplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the five main pathotypes of E. coli, and Shigella spp. This assay will replace the previous molecular genetic methods used in our laboratory and work as an important supplement to the more time-consuming phenotypic assays.
开发了一种多重聚合酶链反应（PCR）检测方法，可在单一反应中扩增7个特定毒力基因和1个内部对照基因，以鉴定致泻性大肠杆菌和志贺氏菌属的5种主要致病型。为每种类别选择的毒力基因如下：肠出血性大肠杆菌（EHEC）的志贺毒素1（Stx1）、志贺毒素2（Stx2）和紧密黏附素（eaeA）；肠致病性大肠杆菌（EPEC）的eaeA；产肠毒素大肠杆菌（ETEC）的耐热肠毒素B亚单位（STIb）和不耐热肠毒素（LTI）；肠侵袭性大肠杆菌（EIEC）和志贺氏菌属的侵袭性质粒抗原H（ipaH）；以及聚集性大肠杆菌（EAEC）的聚集性黏附菌毛调节基因（aggR）。每个正向引物都用荧光染料标记，PCR产物在ABI PRISM310遗传分析仪（应用生物系统公司）上通过多色毛细管电泳进行分离。如果存在，可鉴定出每个毒力基因的多个基因变体。在所分析的110株临床菌株中均扩增出了编码16S核糖体RNA的内部对照基因rrs。在这些菌株中有103株（94%）检测到了毒力基因。在大多数情况下（98/103，95%），通过新型多重PCR检测方法获得的分类与先前通过表型检测结合其他分子遗传学方法确定的分类一致。已经发表了许多多重PCR检测方法，但其中只有少数几种能在单一反应中检测出所有5种大肠杆菌致病型，而且没有一种使用多色毛细管电泳来分离PCR产物。本文介绍的八重PCR检测方法结合毛细管电泳提供了一种简单、可靠且快速的程序，可在单一反应中鉴定大肠杆菌和志贺氏菌属的5种主要致病型。该检测方法将取代我们实验室以前使用的分子遗传学方法，并作为耗时更长的表型检测的重要补充。
