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Intra- and inter-laboratory evaluation of an improved multiplex-PCR method for detection and typing of Salmonella

Martínez Ballesteros, Ilargi and Paglietti, Bianca and Rementeria, Aitor and Laorden, Lorena and Garcia-Ricobaraza, Maria and Bikandi, Joseba and Rubino, Salvatore and Garaizar, Javier (2012) Intra- and inter-laboratory evaluation of an improved multiplex-PCR method for detection and typing of Salmonella. The Journal of Infection in Developing Countries, Vol. 6 (5), p. 443-451. eISSN 1972-2680. Article.

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Abstract

Introduction: We developed and evaluated a multiplex-PCR method for rapid detection of the most common Salmonella serovars in both developed and developing countries. Additionally, the stability of the premixed reagents at high room temperature was studied.
Methodology: Fifty-two Salmonella strains belonging to the collections of the University of Sassari, Italy, and to the University of the Basque Country, Spain, and a collection of a hundred blinded strains, were used to evaluate the multiplex-PCR. Primers targeting genes STY1599 and fliC were selected, and the method was evaluated both intra and inter-laboratories.
Results: The inter-laboratory reproducibility was 95.92%, with a kappa index of 0.757 that indicates a substantial agreement and a high accuracy (80.81%). The sensitivity, specificity, accuracy and precision indexes for the Salmonella genus and S. Typhi targets were maximum, although the targets for Paratyphi A, Typhimurium and Enteritidis showed less accuracy. During a seven-week period, hot-start multiplex-PCR runs were performed with reagents mixed with wax to test their stability at 30°C, and no significant variation in the patterns of amplification was observed.
Conclusions: An improved multiplex-PCR for rapid detection of the most common serovars of Salmonella operable in both developed and developing countries has been designed and tested intra and inter-laboratories. Following a careful optimization protocol will not only allow the confirmation of any suspicious colony by the amplification of the Salmonella genus target, but also the preliminary adscription to the prevalent serovars. Premixed reagents with wax facilitate the throughput and stability of reagents at high room temperatures.

Item Type:Article
ID Code:8797
Status:Published
Refereed:Yes
Uncontrolled Keywords:Salmonella, multiplex-PCR, Typhi, Paratyphi A, enteritidis, typhimurium
Subjects:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
Divisions:001 Università di Sassari > 01-a Nuovi Dipartimenti dal 2012 > Scienze Biomediche
Publisher:OLoeP
eISSN:1972-2680
Copyright Holders:© 2012 Bentancor et al.
Deposited On:18 Mar 2013 10:03

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