Real-time PCR for identification of spp: a comparative study of IS, and target genes
Résumé
Culture is considered as the reference standard assay for diagnosis of 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: () the insertion sequence IS, () and () genes for the detection of 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 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 ovis, bv. 1, bv. 1 and reference strains. Finally, intra-assay repeatability and inter-assay reproducibility were assessed. All species DNA were amplified in the three tests. However, the earliest signal was observed with the IS real-time PCR, where it varied according to the IS 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 IS 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 IS-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 .
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