Tobler Nadia E, Pfunder Monika, Herzog Katrin, Frey Juerg E, Altwegg Martin
Institute of Medical Microbiology, University of Zurich, CH-8028 Zurich, Switzerland.
J Microbiol Methods. 2006 Jul;66(1):116-24. doi: 10.1016/j.mimet.2005.10.016. Epub 2005 Dec 19.
Infections with mycobacteria are an important issue in public health care. Here we present a "proof-of-principle" concept for the identification of 37 different Mycobacterium species using 5' exonuclease real-time PCR and DNA microarray based on the region upstream of the 65 kDa heat shock protein. With our two PCR probes, one complementary to all mycobacteria species, the other specific for the M. tbc-complex, 34 species were properly classified by real-time PCR. After reamplification and hybridization to a DNA microarray, all species showed a specific pattern. All 10 blindly tested positive cultures revealed a positive real-time PCR signal with the genus probe. After reamplification and hybridization, six samples could unambiguously be identified. One sample showed a mixture of presumably three species-specific patterns and sequencing the 16S rRNA confirmed the presence of a mixture. The hybridization results of three specimens could not be interpreted because the signal to background ratio was not sufficient. Two samples considered as negative controls (LAL Reagent Water (Cambrex) and DNA of Candida albicans) gave neither a genus nor a M. tbc-complex positive PCR signal. Based on these results we consider our method to be a promising tool for the rapid identification of different mycobacteria species, with the advantage of possible identification of mixed infections or contaminations.
分枝杆菌感染是公共卫生保健中的一个重要问题。在此，我们提出一种“原理验证”概念，用于基于65 kDa热休克蛋白上游区域，使用5'核酸外切酶实时PCR和DNA微阵列鉴定37种不同的分枝杆菌物种。使用我们的两种PCR探针，一种与所有分枝杆菌物种互补，另一种对结核分枝杆菌复合群特异，通过实时PCR对34个物种进行了正确分类。在重新扩增并与DNA微阵列杂交后，所有物种都显示出特定模式。所有10个经盲法测试的阳性培养物在用属特异性探针检测时均显示出阳性实时PCR信号。在重新扩增并杂交后，6个样本能够明确鉴定。一个样本显示出可能由三种物种特异性模式组成的混合物，对16S rRNA测序证实存在混合物。三个样本的杂交结果无法解释，因为信号与背景的比率不足。两个被视为阴性对照的样本（LAL试剂水（Cambrex）和白色念珠菌DNA）既未给出属特异性PCR阳性信号，也未给出结核分枝杆菌复合群特异性PCR阳性信号。基于这些结果，我们认为我们的方法是快速鉴定不同分枝杆菌物种的一种有前景的工具，具有能够鉴定混合感染或污染的优势。
