Deepa P, Therese K L, Madhavan H N
L and T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
Indian J Med Res. 2005 May;121(5):694-700.
Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level.
The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests.
PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum.
PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.
传统的分枝杆菌鉴定方法是通过标准生化试验来完成的，这些试验耗时、费力且结果并非总是确凿无疑。因此，本研究旨在标准化一种基于聚合酶链反应的简单、快速且经济高效的限制性片段长度多态性分析（PCR-RFLP）方法，该方法使用编码16S - 23S rRNA间隔区的引物，将分枝杆菌分离株鉴定到种水平。
使用标准分枝杆菌菌株对靶向16S-23S rRNA间隔区的引物进行PCR标准化，并应用于51株临床分离株。PCR扩增产物使用限制性内切酶Hae III、MspI和BstXI进行RFLP分析。将获得的结果与传统生化试验的结果进行比较。
PCR能够灵敏地检测到2.5 pg的H37Rv DNA（慢生长分枝杆菌为370 bp）和1.5 pg的偶发分枝杆菌DNA（快生长分枝杆菌为450 bp）。根据获得的PCR-RFLP产物，51株分枝杆菌分离株被分为41株慢生长菌和10株快生长菌。在41株慢生长菌中，40株被鉴定为结核分枝杆菌，1株为蟾分枝杆菌，10株快生长菌为偶发分枝杆菌。
使用靶向16S-23S rRNA间隔区引物的PCR是一种可靠的工具，可在分离后4小时内快速将分枝杆菌分离株鉴定为慢生长菌和快生长菌，并在6-8小时内通过PCR-RFLP进一步进行菌种鉴定。
