不同分子生物学方法快速鉴别非结核分枝杆菌的应用评价

The evaluation of different molecular methods for rapid identification of non-tuberculous Mycobacterium

目的评价3种分子生物学方法快速鉴定非结核分枝杆菌的优缺点。方法收集41株临床分离的非结核分枝杆菌，以16SrRNA基因测序方法为标准，同时以hsp65基因测序方法及PCR—RFLP方法鉴定菌株，与16SrRNA基因测序结果进行比较。结果41株非结核分枝杆菌16SrRNA基因测序结果：9株龟分枝杆菌复合群，7株偶发分枝杆菌，7株胞内分枝杆菌，3株鸟分枝杆菌，3株堪萨斯分枝杆菌复合群，3株耻垢分枝杆菌，3株土分枝杆菌，2株草分枝杆菌，2株无色分枝杆菌，1株瘰疬分枝杆菌，1株M.arupense。与16SrRNA基因测序相比较，hsp65PCR—RFLP能鉴定9株龟分枝杆菌复合群至亚种脓肿分枝杆菌，3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌；1株偶发分枝杆菌及l株无色分枝杆菌与其不符；其余菌株鉴定结果一致，符合率为95．1％（39／41）。hsp65基因测序结果显示，1株爱尔兰分枝杆菌与16SrRNA测序结果不符，其余菌株鉴定结果与其一致，符合率为97．6％（40／41），并且能进一步将9株龟分枝杆菌复合群鉴定至亚种脓肿分枝杆菌，3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌。结论3种方法均能快速鉴定非结核分枝杆菌。与16SrRNA基因测序相比，hsp65基因测序及hsf）65PCR—RFLP更容易鉴定临床最常见非结核分枝杆菌（如堪萨斯分枝杆菌和脓肿分枝杆菌），可在临床推广使用。

Objective To evaluate three molecular methods for rapid identification of non- tuberculous Mycobacterium （NTM）. Methods Forty-one clinical NTM isolates were collected and 16S rRNA gene sequencing was used as the standard method for NTM identification. Meanwhile, the restriction fragment length polymorphism of hsp65 PCR-RFLP and hsp65 gene sequencing were used to identify NTM strains and compared with 16S rRNA gene sequencing. Results The results of 16S rRNA sequencing showed that there were nine Mycobacterium chelonae complex strains, seven Mycobacteriumfortuitum strains, seven Mycobacterium intracellulare strains, three Mycobacterium avium strains, three Mycobacterium kansasii complex strains, three Mycobacterium smegrnatis strains, three Mycobacterium terrae strains, two Mycobacterium phlei strains, two Mycobacterium nonchromogenicum strains, one Mycobacterium scrofulaceum strain and one Mycobacterium arupense strain. Compared with 16S rRNA gene sequencing, hsp65 PCR-RFLP could identify nine Mycobacterium chelonae complexes and three Mycobacterium kansasii complexes to subspecies Mycobacterium abscessus and Mycobacterium kansasii, respectively; One Mycobacterium fortuitum strain and one Mycobacterium nonchromogenicum strain were different from 16S rRNA gene sequencing results , but other isolates were the same. The coincidence was 95.1%. By hsp65 gene sequencing, only one identification of Mycobacterium hiberniae strain was different from 16S rRNA gene sequencing and the coincidence was 97.6%. And hsp65 gene sequencing could further identify nine Mycobacterium chelonae complexes and three Mycobacterium kansasii complexes to subspecies Mycobacterium abscess and Mycobacterium kansasii, respectively. Conclusions All three molecular methods can identify NTM strains rapidly. Compared with 16S rRNA gene sequencing, hsp65 gene sequencing and hsp65 PCR-RFLP are easier to identify clinical common NTM strains （such as Mycobacterium kansasii and Mycobacterium abscessus） , and can be widely used in clinical practice.