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Author manuscript, published in "Veterinary Microbiology 137, 1-2 (2009) 156" DOI : 10.1016/j.vetmic.2008.12.023

Accepted Manuscript
Title: Real-time PCR for identification of Brucella spp: a comparative study of IS711, bcsp31and per target genes Authors: Lotfi Bounaadja, David Albert, Benoˆt Ch´ nais, ı e Sylvie H´ nault, Michel S. Zygmunt, Sylvie Poliak, Bruno e Garin-Bastuji PII: DOI:Reference: S0378-1135(08)00609-3 doi:10.1016/j.vetmic.2008.12.023 VETMIC 4313 VETMIC 18-7-2008 22-12-2008 29-12-2008

peer-00485525, version 1 - 21 May 2010

To appear in: Received date: Revised date: Accepted date:

Please cite this article as: Bounaadja, L., Albert, D., Ch´ nais, B., H´ nault, S., Zygmunt, e e M.S., Poliak, S., Garin-Bastuji, B., Real-time PCR for identification of Brucella spp:a comparative study of IS711, bcsp31and per target genes, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2008.12.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it ispublished in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Real-time PCR for identification of Brucella spp: a comparative study of IS711, bcsp31and per target genes

Lotfi Bounaadja 1,2, David Albert3, Benoît Chénais1, Sylvie Hénault3, Michel S.Zygmunt4, Sylvie Poliak2, and Bruno Garin-Bastuji3.

Santé, 72085 Le Mans, France.
2

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Laboratoire Départemental de la Sarthe, 72000, Le Mans, France.

3

OIE/FAO and EU Community Reference Laboratory for Brucellosis, French Food Safety Agency

(AFSSA), 94706, Maisons-Alfort, France.
4

UR1282,Infectiologie Animale et Santé Publique (IASP), Institut National de la Recherche

Agronomique (INRA), 37380, Nouzilly, France

Corresponding author:

Bruno Garin-Bastuji, AFSSA, OIE/FAO and EU Community Reference Laboratory for Brucellosis, 23 avenue du Général de Gaulle, 94706 Maisons-Alfort Cedex, France. Tel. : + 33 1 49 77 13 00 - Fax : + 33 1 49 77 13 44 - E-mail :b.garin-bastuji@afssa.fr

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Université du Maine, Laboratoire de Biologie et Génétique Evolutive, EA2160 Mer Molécules

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ABSTRACT Culture is considered as the reference standard assay for diagnosis of Brucella spp. in humans and animals but it is time-consuming and hazardous. In this study, we evaluated theperformances of newly designed real-time PCR assays using Taqman® probes and targeting the 3 following specific genes: (i) the insertion sequence IS711, (ii) bcsp31 and (iii) per genes for the detection of Brucella at genus level. The real-time PCR assays were compared to previously described conventional PCR assays targeting the same genes. The genus-specificity was evaluated on 26 Brucella strains,clinically relevant, phylogenetically related or serologically cross-reacting micro-organisms. The analytical sensitivity was assessed using decreasing DNA quantities of Brucella ovis, B. melitensis bv. 1, B. abortus bv. 1 and B. canis reference strains. Finally, intra-assay repeatability and interassay reproducibility were assessed. All Brucella species DNA were amplified in the three tests.However, the earliest signal was observed with the IS711 real-time PCR, where it varied according to the IS711 copy number. No cross-reactivity was observed in all three tests. Real-time PCR was always more sensitive than conventional PCR assays. The real-time PCR assay targeting IS711

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was very low. In...