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Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes

Bounaadja, Lotfi, Albert, David, Chénais, Benoît, Hénault, Sylvie, Zygmunt, Michel S., Poliak, Sylvie, Garin-Bastuji, Bruno
Veterinary microbiology 2009 v.137 no.1-2 pp. 156-164
zoonoses, Brucella, brucellosis, disease detection, disease diagnosis, polymerase chain reaction, new methods, rapid methods, DNA probes, genes, transposons, ligases, molecular epidemiology, pathogen identification, validity, accuracy, phylogeny, molecular genetics
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 the performances 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, including all species and biovars. The analytical specificity was evaluated on a collection of 68 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 inter-assay 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 presented an identical or a greater sensitivity than the two other tests. In all cases, the variability was very low. In conclusion, real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella.