鉴别结核分枝杆菌复合群的新型核酸扩增检测靶标评价

Identification and evaluation of a new nucleic acid amplification test target for specific detection ofMycobacterium tuberculosis complex

目的寻找新的鉴别诊断MTB复合群（MTC）高特异度和敏感的核酸靶标。方法利用简单序列重复区间（ISSR）分型技术平台筛选MTC的特征片段，克隆测序获得特征序列并进行序列同源性分析，以该序列为基础设计MTC特征引物，并对211株分枝杆菌菌株（其中MTC107株、非结核分枝杆菌104株）进行鉴别检测。利用分枝杆菌属特征序列16s rRNA和MTC特征序列IS6110的鉴别结果，对MTC特征引物检测结果进行评估。结果通过ISSR分型获得588bp的MTC特征片段，该序列为MTC菌株的特征序列，位于MTB标准株H37Rv基因组的315947-316534位，以该序列为基础设计MTC鉴别引物MTCF／R，16srRNA基因在211株分枝杆菌菌株中的扩增结果均为阳性，IS6110序列在107株MTC菌株中扩增阳性率为99％（106／107），在104株NTM菌株中阴性检出率为100％（104／104），MTCF／R引物在MTC菌株中的扩增阳性率为100％（107／107），在非结核分枝杆菌菌株中的阴性检出率为100％（104／104）。结论通过ISSR分型技术筛选出的MTC特征序列可作为新的核酸扩增靶标用于MTC和非结核分枝杆菌的鉴别诊断。

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 mycobaeterial 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 Myeobacterium） 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 I^7 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.