Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

被引：0
作者：
Elodie Barbau-Piednoir
Nadine Botteldoorn
Marc Yde
Jacques Mahillon
Nancy H. Roosens
机构：
[1] Scientific Institute of Public Health,Faculty of Bioscience Engineering, Earth and Life Institute
[2] Université Catholique de Louvain,undefined
来源：
Applied Microbiology and Biotechnology | 2013年 / 97卷
关键词：
Real-time PCR; SYBR®Green; Foodborne pathogens; Detection; qPCR validation;
D O I：
暂无
中图分类号：
学科分类号：
摘要：
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).
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页码：4021 / 4037
页数：16
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