基于PCR熔解曲线技术的结核分枝杆菌Spoligotyping基因分型的临床应用研究  

Clinical application of spoligotyping based on PCR fusion curve technology in Mycobacterium tuberculosis genotypes

作　　者：蓝如束 叶婧[2] 罗丹 覃慧芳[2] 黄莉雯[2] 苏华斌 罗淋尹 林玫[2] LAN Ru-shu;YE Jing;LUO Dan;QIN Hui-fang;HUANG Li-wen;SU Hua-bin;LUO Lin-yin;LIN Mei(Jiangbin Hospital,Nanning 530021,China;Department of Tuberculosis Control,Guangxi Zhuang Autonomous Region Center for Disease Control and Prevention,Nanning 530028,China;Department of Biostatistics,Public Health and Management,Guangxi University of Chinese Medicine,Nanning 530200,China)

机构地区：[1]广西壮族自治区江滨医院,南宁530021 [2]广西壮族自治区疾病预防控制中心,南宁530028 [3]广西中医药大学公共卫生与管理学院生物统计学教研室,南宁530200

出　　处：《中国人兽共患病学报》2021年第4期285-291,328,共8页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.81560549);广西自然科学基金(No.2017GXNSFAA198330)联合资助。

摘　　要：目的利用基于PCR熔解曲线技术的结核分枝杆菌Spoligotyping基因分型方法(McSpoligotyping)对结核分枝杆菌分离株进行基因分型,以评价其在结核病流行病学调查中的应用价值。方法对广西2017年结核耐药监测点收集的949株结核分枝杆菌,采用McSpoligotyping分型方法对其进行基因分型。首先,通过对其中的361株菌株的McSpoligotyping分型结果与全基因组测序(WGS)结果的一致性比较来评价McSpoligotyping分型方法。其次,对949株菌株分型数据与SITVITWEB数据库进行比对,获取间隔区寡核苷酸国际型别(spoligotype intemational type,SIT)编号,将分型结果提交到https://www.miru-vntrplus.org/MIRU/index.faces网站进行聚类分析。结果McSpoligotyping分型与WGS结果一致率为94.18%(340/361),在结果不一致标本中,出现不一致较多间隔序列在第5间隔。在949株菌株中新发现基因型为121株,已定义基因型为828株;其中已定义基因型共分为84种基因型,主要归属于5种基因家族(BEIJING、T、H、EAI、LAM)和3个家系(Lineage 1、Lineage 2、Lineage 4);5种基因家族主要流行为BEIJING和T基因家族,家系中主要流行为Lineage 2。949株菌株中的822株聚为49个簇(2~528株),最大一簇为Beijing家族SIT1基因型,其余的127株菌表现为独特的基因型。结论利用PCR熔解曲线技术可快速对MTB进行基因分型,此技术较传统方法操作简便、耗时短,对结核病疫情处置及结核病分子流行病学研究具有重要意义。This study aimed to evaluate the value of spoligotyping based on PCR melting curve technology(McSpoligotyping)for Mycobacterium tuberculosis(MTB)genetic typing in the investigation of tuberculosis epidemiology.A total of 949 MTB strains collected from drug resistance monitoring sites in 2017 in Guangxi province were genotyped with McSpoligotyping.The genetic typing results of 361 strains were compared with the sequences from whole genome sequencing(WGS)for McSpoligotyping assessment.The typing data of 949 strains were compared with those in the SITVITWEB database,and the spoligotype intemational type(SIT)numbers were obtained from the SITVITWEB database.The genetic typing results were submitted to https://www.miru-vntrplus.org/MIRU/index.faces for cluster analysis.The coincidence rate of the results(McSpoligotyping and WGS)was 94.18%(340/361).Among the inconsistent specimens,the interval sequences with more inconsistencies were found in 5th.Among the 949 strains’genotypes,121 were newly discovered,and 828 had been defined.The 828 defined genotypes were classified into 84 genotypes,which mainly belonged to five gene families(BEIJING,T,H,EAI and LAM)and three lineages(lineage 1,lineage 2 and lineage 4).The BEIJING and T gene families,and lineage 2 were the major genotypes.Of the 949 strains,822 were clustered into 49 clusters(2 to 528 strains).The largest cluster was the Beijing family SIT1 genotype,and the remaining 127 strains showed unique genotypes.MTB can be genotyped quickly with PCR melting curve technology,which is simpler and less time-consuming than traditional methods.This method should be highly useful in the management of tuberculosis epidemics and molecular epidemiological research.

关 键 词：结核分枝杆菌 McSpoligotying PCR熔解曲线技术 WGS 

分 类 号：R378.91[医药卫生—病原生物学]