Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes
Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes
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DOI：
10.1016/j.vetmic.2008.12.023
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发表时间：
2009-05-28
影响因子：
3.3
通讯作者：
Garin-Bastuji, Bruno
中科院分区：
文献类型：
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作者：
Bounaadja, Lotfi;Albert, David;Garin-Bastuji, Bruno
在人类和动物中，培养被视为诊断布鲁氏菌属的参考标准检测方法，但它耗时且具有危险性。在本研究中，我们评估了新设计的使用TaqMan®探针并针对以下3个特定基因的实时荧光定量PCR检测方法的性能：（i）插入序列IS711，（ii）bcsp31以及（iii）per基因，用于在属水平上检测布鲁氏菌。将实时荧光定量PCR检测方法与先前描述的针对相同基因的常规PCR检测方法进行了比较。对26株布鲁氏菌菌株（包括所有种和生物型）评估了属特异性。对68种临床相关的、系统发育相关的或血清学交叉反应的微生物集合评估了分析特异性。使用递减数量的羊布鲁氏菌、马尔他布鲁氏菌生物型1、牛布鲁氏菌生物型1和犬布鲁氏菌参考菌株的DNA评估了分析灵敏度。最后，评估了批内重复性和批间重现性。在这三种检测中，所有布鲁氏菌属的DNA都被扩增。然而，在IS711实时荧光定量PCR中观察到最早的信号，且该信号根据IS711拷贝数而变化。在所有三种检测中均未观察到交叉反应。实时荧光定量PCR总是比常规PCR检测方法更灵敏。针对IS711的实时荧光定量PCR检测方法比其他两种检测方法具有相同或更高的灵敏度。在所有情况下，变异性都非常低。总之，实时荧光定量PCR检测方法易于使用，比常规PCR系统更快地产生结果，同时降低了DNA污染的风险。基于IS711的实时荧光定量PCR检测方法具有特异性和高灵敏度，是一种快速、安全检测布鲁氏菌属的高效且可重现的方法。（C）2008爱思唯尔B.V.保留所有权利。
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 (R) 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. (C) 2008 Elsevier B.V. All rights reserved.