Laboratoire Eau Environnement Systèmes Urbains (Leesu) UMR MA 102-AgroParisTech, Université Paris-Est, 6-8 avenue Blaise Pascal Cité, Descartes, FR 77455, Champs sur Marne, France.
BMC Microbiol. 2013 Dec 3;13:277. doi: 10.1186/1471-2180-13-277.
The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets.
Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples.
The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.
环境可能是许多致病性分枝杆菌的来源，但通过细菌学工具检测分枝杆菌通常较为困难且耗时。因此，已经提出了几种基于管家基因、非功能 RNA 和结构核糖体 RNA 序列的分子靶标，用于在临床或环境样本中检测和鉴定分枝杆菌。虽然其中一些靶标被提议为该属的特异性靶标，但大多数在包含相关但不同的细菌属的复杂环境样本中容易出现假阳性结果。如今，测序基因组数量的增加以及用于基因组比较的软件的可用性为开发新的、分枝杆菌特异性的靶标以及相关的分子探针和引物提供了工具。因此，我们进行了一次计算机搜索，以寻找仅存在于结核分枝杆菌 H37Rv 基因组中的蛋白质，以便设计敏感和特异的分子靶标。
在结核分枝杆菌 H37Rv 的 3989 个预测蛋白质中，只有 11 个蛋白质与分枝杆菌属的基因组具有 80%到 100%的相似性，与密切相关的棒状杆菌、诺卡氏菌和红球菌属的基因组的相似性小于 50%。基于 DNA 序列比对，我们设计了引物对和探针，特异性检测分枝杆菌的 atpE 基因，这通过对一系列分枝杆菌和非分枝杆菌的定量实时 PCR 得到验证。我们开发的实时 PCR 方法成功地用于检测自来水中和湖泊样品中的分枝杆菌。
结果表明，该针对 atpE 基因的实时 PCR 方法可用于环境样本中分枝杆菌的高度特异性检测和精确定量。
