Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: Optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis
Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: Optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis
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DOI：
10.1128/jcm.42.1.453-457.2004
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发表时间：
2004-01-01
影响因子：
9.4
通讯作者：
Yadav, JS
中科院分区：
文献类型：
--
作者：
Khan, IUH;Yadav, JS
建立了一种单管PCR方法，用于对3111例非结核分枝杆菌（NTM）及其环境分离株进行快速鉴定。优化或开发了以下三个步骤：（i）简单的6分钟直接细胞裂解方案，作为用于产生DNA模板的PCR预步骤，（ii）改进的分枝杆菌特异性PCR扩增方案，其具有更宽的物种特异性，使用新设计的靶向65-kDa节拍休克蛋白（hsp）基因的228-bp区域的引物和最佳PCR扩增条件，和（iii）PCR产物的属特异性限制性分析，以确定性鉴定未知NTM分离物。
A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 111 without conventional DNA isolation. The following three steps were optimized or developed: (i) a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii) an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa beat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii) a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.