Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis
Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis
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开发用于鉴定鼠伤寒沙门氏菌和肠炎沙门氏菌的新型多重 PCR 检测方法
DOI：
10.1016/j.foodcont.2012.01.062
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发表时间：
2012-09-01
期刊：
影响因子：
6
通讯作者：
Shi, Xianming
中科院分区：
文献类型：
--
作者：
Liu, Bin;Zhou, Xiujuan;Shi, Xianming
采用比较基因组方法，分别验证了 38 个和 8 个片段作为鼠伤寒沙门氏菌和肠炎沙门氏菌的特异性鉴定靶点。引物组是根据这些序列设计的，并通过 PCR 测定进行评估。分别针对鼠伤寒沙门氏菌和肠炎沙门氏菌选择了两对针对STM4495和SEN1392基因的高特异性引物，并基于这两对引物和针对肠沙门氏菌特异性srfC基因的另一对引物，建立了多重PCR方法来鉴定这些细菌。多重PCR还包括内部扩增对照(IAC)，其由原壳小球藻的psy基因构建，用于过程控制以监测潜在的PCR抑制剂。对于鼠伤寒沙门氏菌和肠炎沙门氏菌，该多重 PCR 的灵敏度分别为每次 PCR 89 fg 和 138 fg DNA。对于两种血清型的纯培养物，每次 PCR 的检测限低至 22-23 CFU。此外，在37℃富集12小时后，最初被2-3 CFU沙门氏菌污染的牛奶样品中观察到了阳性结果。这些结果表明，比较基因组方法是识别新的特定靶点的有价值的工具，是开发快速、准确的食源性病原体检测方法的必要步骤。 (C) 2012 Elsevier Ltd. 保留所有权利。
Using a comparative genomic method, 38 and 8 fragments were verified as specific identification targets for Salmonella Typhimurium and Enteritidis, respectively. Primer sets were designed based on these sequences and evaluated by PCR assays. Two primer sets targeting the STM4495 and SEN1392 genes with high specificity were selected for S. Typhimurium and Enteritidis, respectively, and a multiplex PCR method was developed for the identification of these bacteria based on these two primer sets and another primer set targeting the srfC gene specific for Salmonella enterica. The multiplex PCR also included an internal amplification control (IAC), which was constructed by psy gene from Chlorella protothecoides for process control to monitor potential PCR inhibitors. The sensitivity of this multiplex PCR was 89 fg and 138 fg of DNA per PCR for S. Typhimurium and S. Enteritidis, respectively. The detection limits were as low as 22-23 CFU per PCR for pure cultures of both serovars. Moreover, positive results were observed from milk samples that were initially contaminated with 2-3 CFU Salmonella after 12 h of enrichment at 37 degrees C. These results demonstrate that the comparative genomic method is a valuable tool for identifying new specific targets, a necessary step for developing rapid and accurate detection methods for foodborne pathogens. (C) 2012 Elsevier Ltd. All rights reserved.