Scientific Institute of Public Health, J. Wytsmanstraat 14, 1050 Brussels, Belgium.
Appl Microbiol Biotechnol. 2013 May;97(9):4021-37. doi: 10.1007/s00253-012-4477-2. Epub 2012 Oct 20.
A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).
本文提出了一种组合使用四种针对两个鉴别水平的定性 SYBR®Green qPCR 筛选检测方法：李斯特菌属（李斯特菌灰色除外）和单核细胞增生李斯特菌。这些检测方法是为了能够使用相同的聚合酶链反应（PCR）程序同时进行而开发的。本文还提出了一种新的验证程序，根据两项准则（ISO 22118 标准和“转基因生物测试分析方法最低性能要求的定义”），专门针对食品微生物学应用的 qPCR 检测方法进行验证。所开发的检测方法针对iap、prs 和 hlyA 基因，这些基因属于李斯特菌属的毒力簇或位于其附近。选择的引物设计用于扩增短片段（60 至 103bp），以获得最佳的 PCR 效率（效率在 97%至 107%之间）。SYBR®Green qPCR 检测方法的检测限为每个 qPCR 反应中目标基因的两个至五个拷贝。这些检测方法具有高度的准确性（李斯特菌属和单核细胞增生李斯特菌检测的准确性分别为 98.08%和 100%）。
