利用改良聚合酶链反应-单链构象多态性技术从临床标本中快速检测耐多药结核分枝杆菌
Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens.
作者信息
Ali Imani Fooladi Abbas, Babak Farzam, Fazlollah Mousavi Seyed, Nematollah Jonaidi Jafari
机构信息
Applied Microbiology, Research Center, Baqiyatallah University Of Medical Sciences, Tehran, Iran.
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
出版信息
Asian Pac J Trop Biomed. 2014 May;4(Suppl 1):S165-70. doi: 10.12980/APJTB.4.2014C1186.
OBJECTIVE
To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.
METHODS
Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.
RESULTS
Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.
CONCLUSIONS
Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.
目的
基于分析katG、rpoB基因的聚合酶链反应-单链构象多态性（PCR-SSCP）技术，设计一种快速检测利福平（RIF）和异烟肼（INH）耐药突变体的试验。
方法
生化试验以及靶向IS6110的PCR显示103份临床样本为结核病。为确定分离株对抗结核药物的敏感性，采用比例法。通过SSCP评估katG、rpoB基因扩增产物中出现的突变。
结果
采用比例法，分别有12株（11.6%）和9株（8.7%）分离株对INH和RIF耐药，9株（8.7%）分离株对两种药物均耐药（耐多药结核病）。通过PCR-SSCP和测序检测到3株（2.9%）耐多药结核病和2株INH耐药分离株。PCR-SSCP对耐多药分离株的敏感性和特异性分别为33%和100%。
结论
SSCP与测序结果完全一致可能表明除我们所关注的基因外，其他基因发生了耐药相关突变。
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