Design and evaluation of a multiplex polymerase chain reaction assay for the simultaneous identification of genes for nine different virulence factors associated with Escherichia coli that cause diarrhea and edema disease in swine
Design and evaluation of a multiplex polymerase chain reaction assay for the simultaneous identification of genes for nine different virulence factors associated with Escherichia coli that cause diarrhea and edema disease in swine
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DOI：
10.1177/104063870902100104
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发表时间：
2009-01-01
影响因子：
1.5
通讯作者：
Bosworth, Brad T.
中科院分区：
文献类型：
--
作者：
Casey, Thomas A.;Bosworth, Brad T.
开发了一种多重聚合酶链反应 (mPCR) 检测方法，用于检测和表征引起猪腹泻和水肿疾病的致病性大肠杆菌。 mPCR 测定被设计为单一反应，用于检测 5 种不同的粘附素（K88、K99、98713、F41 和 1718）、3 种肠毒素（LT、STaP 和 STb）以及与猪致病性大肠杆菌相关的志贺毒素 (Stx2e)。通过与先前对 100 个猪分离株的分析结果进行比较来评估 mPCR 测定的特异性，这些猪分离株的特征是使用 5 种粘附素和 4 种毒素基因的 DNA 探针进行菌落印迹杂交。两种方法之间完全一致。通过检查含有已知具有不同抗原亚型或 DNA 序列变异的毒力因子的菌株，进一步评估了从猪中分离出的大肠杆菌病原体的 mPCR 测定。结果发现，mPCR 检测针对编码 K88 和 F18 的基因，从含有不同 K88 和 F18 抗原亚型基因的菌株中扩增出具有适当大小的产物： mPCR 检测针对编码 STaP 扩增产物的基因，仅来自 STaP 阳性分离株，而非 STaH 阳性分离株； mPCR 检测针对仅来自 Stx2 阳性而非 Stx1 阳性分离株的 Stx2 扩增产物的编码基因。同样，针对 LTI 编码基因的 mPCR 测定并没有从含有 LTII 基因的菌株中产生适当的产物。 mPCR 测定执行起来很简单，并且它们应该可用于诊断猪大肠杆菌病，包括从患有腹泻或水肿疾病的猪中分离出的大肠杆菌的基因型特征。
A multiplex polymerase chain reaction (mPCR) assay was developed for detection and characterization of pathogenic Escherichia coli that cause diarrhea and edema disease in swine. The mPCR assay was designed as a single reaction for detecting 5 different adhesins (K88, K99, 98713, F41, and 1718), 3 enterotoxins (LT, STaP, and STb), and the Shiga toxin (Stx2e) associated with porcine pathogenic E. coli. The specificity of the mPCR assay was evaluated by comparison with results from previous analysis of 100 porcine isolates characterized by colony blot hybridization with DNA probes for the 5 adhesins and 4 toxin genes. There was complete agreement between the 2 methods. The mPCR assay for E coli pathogens isolated from swine was further evaluated by examination of strains containing virulence factors that are known to have different antigenic subtypes or DNA sequence variations. It was found that the mPCR assays targeting genes encoding for K88 and F18 amplified products with the appropriate sizes from strains containing genes for different K88 and F18 antigenic subtypes: mPCR assays targeting the gene encoding for STaP amplified product from only STaP-positive but not STaH-positivec isolates; and mPCR assays targeting the gene encoding for the Stx2 amplified products from only Stx2-positive and not Stx1-positive isolates. Similarly, mPCR assays targeting the gene encoding for LTI did not produce the appropriate product from strains containing genes for LTII The mPCR assays are simple to perform, and they should be useful for diagnosis of porcine colibacillosis, including the genotypic characterization of E coli isolates from pigs with diarrhea or edema disease.