[一种用于结核分枝杆菌复合群特异性检测的新型核酸扩增试验靶点的鉴定与评估]

[Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex].

作者信息

Gao Shi-hui, Zhao Ying-wei, Hu Zhong-yi, Wang Jie, Lu Jun-mei, Yan Li-ping, Guo Qi, Ma Hui, Qin Lian-hua

机构信息

Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China.

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2012 Dec;35(12):907-10.

PMID:23328181

Abstract

OBJECTIVE

To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC).

METHODS

MTC-specific fragment was obtained by ISSR genotyping technology. Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains. IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains.

RESULTS

One MTC-specific fragment with the length of 588 bp, located in 315947 - 316534 of the genome from MTB reference strain H(37) Rv, were obtained, cloned and sequenced. MTC-specific primer pairs MTCF/R were designed based on these sequences. All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon. All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences. All NTM strains were negative in both IS6110 and MTCF/R PCR amplification.

CONCLUSIONS

The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.

摘要

目的

鉴定并评估一种用于特异性检测结核分枝杆菌复合群（MTC）的新型核酸扩增（NAA）检测靶点。

方法

通过ISSR基因分型技术获得MTC特异性片段。基于MTC特异性片段的序列设计引物对，并在211株分枝杆菌菌株中进行检测，其中包括107株MTC菌株和104株非结核分枝杆菌（NTM）菌株。以IS6110元件（用于MTC菌株的特异性鉴定）和16s rRNA基因（用于分枝杆菌的特异性鉴定）扩增作为对照，评估NAA检测靶点在检测MTC菌株中的效能。

结果

获得了一个长度为588 bp的MTC特异性片段，其位于结核分枝杆菌参考菌株H(37)Rv基因组的315947 - 316534处，进行了克隆和测序。基于这些序列设计了MTC特异性引物对MTCF/R。所有211株分枝杆菌菌株均准确产生了属特异性的16s rRNA扩增子。所有MTC菌株在MTCF/R PCR扩增中均为阳性，而99%的MTC菌株（106/107）在IS6110序列扩增中为阳性。所有NTM菌株在IS6110和MTCF/R PCR扩增中均为阴性。