title: Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.
abstract: 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).
authors.affiliation: Scientific Institute of Public Health, J. Wytsmanstraat 14, 1050 Brussels, Belgium. epiednoir@wiv-isp.be
authors.forename: Elodie
authors.initials: E
authors.lastname: Barbau-Piednoir
authors.forename: Nadine
authors.initials: N
authors.lastname: Botteldoorn
authors.forename: Marc
authors.initials: M
authors.lastname: Yde
authors.forename: Jacques
authors.initials: J
authors.lastname: Mahillon
authors.forename: Nancy H
authors.initials: NH
authors.lastname: Roosens
mesh_terms: D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics; D005516:Food Microbiology / Q000379:methods*; D008087:Listeria / Q000235:genetics* / Q000302:isolation & purification; D008089:Listeria monocytogenes / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins