采用 RFLP 和 spoligotyping 方法测定基因型和多重耐药结核分枝杆菌的基因分型。

Determination of genotypic varieties and genotyping of multiple drug-resistant tuberculosis by the RFLP and spoligotyping methods.

机构信息

Department of Medical Microbiology, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey.

Department of Medical Microbiology, Faculty of Medicine, Sanko University, Gaziantep, Turkey.

出版信息

Turk J Med Sci. 2016 Nov 17;46(5):1502-1511. doi: 10.3906/sag-1412-5.

DOI:10.3906/sag-1412-5

PMID:27966321

Abstract

BACKGROUND/AIM: The purpose of the present study was to determine the distribution and epidemiological features of mycobacteria with molecular methods.

MATERIALS AND METHODS

Fifty-five culture-positive samples were analyzed by polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) at species level, and their molecular typing was performed with spoligotyping. The IS6110 region and the locus of gene coding for Hsp65 were amplified. RFLP profiles were obtained by cutting the Hsp65 region with the Hae III and BstE II (Eco91I) enzymes. Spoligotyping was carried out by commercial kit. The H37Rv strain was used as the control.

RESULTS

All samples showed the same cutting pattern with the H37Rv strain. The RFLP profiles of 9 strains identified as "mycobacteria other than tuberculosis" were compatible with the M. tuberculosis complex. Spoligotyping of 55 isolates detected 13 different genetic profiles. The Beijing genotype was not detected. One isolate was described as an orphan strain according to the SpolDB4 database. The most frequently detected family was T1 with 32 strains (64%), followed by 9 isolates (18%) belonging to the LAM7 TUR family.

CONCLUSION

PCR-RFLP is a specific, rapid, and effective method in routine diagnosis of mycobacteria. Spoligotyping is an ideal method in the determination of genotypic varieties of mycobacteria.

摘要

背景/目的：本研究旨在通过分子方法确定分枝杆菌的分布和流行病学特征。

材料和方法

采用聚合酶链反应-限制性内切酶长度多态性（PCR-RFLP）对 55 份培养阳性样本进行种水平分析，并进行 spoligotyping 分子分型。扩增 IS6110 区和编码 Hsp65 基因的位点。用 Hae III 和 BstE II（Eco91I）酶切割 Hsp65 区获得 RFLP 图谱。 spoligotyping 采用商业试剂盒进行。使用 H37Rv 株作为对照。

结果

所有样本均与 H37Rv 株表现出相同的切割模式。9 株鉴定为“非结核分枝杆菌”的菌株的 RFLP 图谱与结核分枝杆菌复合体相符。55 株分离株 spoligotyping 检测到 13 种不同的遗传图谱。未检测到北京基因型。根据 SpolDB4 数据库，一株分离株被描述为孤儿株。最常见的家族是 T1，有 32 株（64%），其次是 LAM7 TUR 家族的 9 株（18%）。