High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis
High-Throughput Molecular Determination of Salmonella enterica Serovars by Use of Multiplex PCR and Capillary Electrophoresis Analysis
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DOI：
10.1128/jcm.02095-08
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发表时间：
2009-05-01
影响因子：
9.4
通讯作者：
Boyle, David S.
中科院分区：
文献类型：
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作者：
Leader, Brandon T.;Frye, Jonathan G.;Boyle, David S.
肠道沙门氏菌是世界范围内食源性疾病的主要原因，也是家畜和野生动物发病和死亡的主要原因。在本研究中，开发了一种高通量分子检测方法来确定最常见的临床和非人血清型的S。在美国的enterica。根据其在常见血清型中的差异分布，鉴定了16个基因组靶标。设计引物以在单个多重PCR中扩增这些靶中的每一个的区域，同时掺入6-羧基荧光素标记的通用引物以荧光标记所有扩增子。使用毛细管电泳分离荧光标记的PCR产物，并基于每个靶基因产生的PCR产物的存在或不存在，为每个分离株产生用于快速分型（SMART）代码的沙门氏菌多重测定。2007年收集了来自华盛顿州的751株沙门氏菌盲法临床分离株，之前通过抗血清进行了血清分型，使用该检测方法进行了筛选。根据与先前确定的美国前50种最常见血清型的一组代表性SMART代码的比较，共正确鉴定了89.6%的分离株。在其余分离株中，6.2%代表为先前确定的血清型产生新SMART代码的分离株，而最终8.8%来自未在设盲研究中用于分离株评分的原始样本组中筛选的血清型。这种高通量的多重PCR检测允许简单和准确的分型最流行的临床血清型的肠道沙门氏菌的水平与传统的血清分型，但在一小部分的成本和时间所需的每个测试。
Salmonella enterica is a leading cause of food-borne illness worldwide and is also a major cause of morbidity and mortality in domestic and wild animals. In the current study, a high-throughput molecular assay was developed to determine the most common clinical and nonhuman serovars of S. enterica in the United States. Sixteen genomic targets were identified based on their differential distribution among common serovars. Primers were designed to amplify regions of each of these targets in a single multiplex PCR while incorporating a 6-carboxyfluorescein-labeled universal primer to fluorescently label all amplicons. The fluorescently labeled PCR products were separated using capillary electrophoresis, and a Salmonella multiplex assay for rapid typing (SMART) code was generated for each isolate, based upon the presence or absence of PCR products generated from each target gene. Seven hundred fifty-one blind clinical isolates of Salmonella from Washington State, collected in 2007 and previously serotyped via antisera, were screened with the assay. A total of 89.6% of the isolates were correctly identified based on comparison to a panel of representative SMART codes previously determined for the top 50 most common serovars in the United States. Of the remaining isolates, 6.2% represented isolates that produced a new SMART code for a previously determined serotype, while the final 8.8% were from serotypes not screened in the original panel used to score isolates in the blinded study. This high-throughput multiplex PCR assay allowed simple and accurate typing of the most prevalent clinical serovars of Salmonella enterica at a level comparable to that of conventional serotyping, but at a fraction of both the cost and time required per test.