中国麻风分枝杆菌种型分布与特征

Distribution and characteristics on species of Mycobacterium leprae in China

目的了解中国目前传播的麻风分枝杆菌(M.leprae)菌种及单核苷酸多态性(SNPs)型分布与特征。方法对来自中国22个省市171例麻风患者皮损组织样本,采用巢式PCR扩增麻风分枝杆菌特异16S rRNA保守片段,并对PCR阳性产物直接测序,经BLAST进行序列比对;采用PCR产物限制性酶切基因多态性方法对麻风分枝杆菌进行SNP分型。结果 171例标本扩增片段与来自巴西的麻风分枝杆菌Br4923相似性达99%,均为M.leprae,未检出M.lepromatosis。在85例SNP分型标本中,SNP3型、SNP1型和SNP2型分别占78.8%(67/85)、20%(17/85)和1.2%(1/85),未检出SNP4型。130例16S rRNA序列存在C251T位碱基变异,不同临床型别标本中16S rRNA序列突变及SNP型别分布差异无统计学意义;16S rRNA基因C251T突变与麻风分枝杆菌菌株的SNP分型有一定的关联,存在突变的菌株多为SNP3型,少见SNP1型,未见SNP2型;不同地区的SNP型别分布之间差异有统计学意义,内陆地区的SNP3型菌株分布率97.1%(34/35)显著高于沿海66%(33/50)(χ~2=11.96,P<0.01)。不同地区的16S rRNA序列突变率差异也有统计学意义,内陆地区的16S rRNA突变率94.8%(92/97)显著高于沿海51.4%(38/74)(χ~2=43.56,P<0.01)。结论麻风分枝杆菌16S rRNA基因序列突变C251T与SNP分型有关,可提示不同基因型麻风分枝杆菌的地理分布。未发现麻风分枝杆菌M.lepromatosis菌种。

Objective：To realize the distribution and characteristics of Mycobacterium leprae （M.leprae） species and its single nucleotide polymorphisms （SNPs） spreading currently in China. Methods：A total of 171 cutaneous lesion specimen of leprosy patients from 22 provinces were collected. The 16S rRNA conservative region of Mycobacterium leprae was amplified by nest PCR and the positive products were sequenced directly and aligned by BLAST. The SNPs of M.leprae were genotyped by restricted fragment length polymorphism for the PCR products. Results：The 171 specimen were all Mycobacterium leprae since the amplified fragments of DNA samples were 99% similar to the Br4923 of M.leprae from Brazil. No new species （M. lepromatosis） was found. Among the 85 samples genotyped for SNPs, SNP3, SNP1 and SNP2 accounted for 78.8% （67/85）, 20% （17/85） and 1.2% （1/85） respectively. There was no sample with SNP4 genotype to be detected. Among the 171 sequencing specimen, 130 showed mutation C T at 251 bp of 16S rRNA. There was no difference for mutant rate of 16S rRNA gene and SNP genotype among the samples with different clinical pathological types. Certain associations between 16S rRNA C251T mutation and SNP genotype were found. Most of the samples with C251T mutations of 16S rRNA sequence were SNP3, only a few were SNP1 but not SNP2. There was significant difference of SNP genotype distribution among the patients from different regions. The distribution rate of SNP3 genotype in the samples from inland region （97.1%, 34/35） was significantly higher than that from coastal region （66%, 33/50） （χ2=11.96,P〈0.01）.There was significant difference of the gene mutation rate of 16S rRNA sequence among the patients from different regions. The mutation rate of 16S rRNA in the samples from inland region （94.8%, 92/97） was significantly higher than that from coastal region （51.4%, 38/74） （χ2=43.56,P〈0.01）. Conclusion：C251T mutation in 16S rRNA gene sequence of M.leprae may associate with SNP type suggesting that the characteristics of geographical distribution presented in different genotypes of M.leprae. No new species of M.leprae was found in this study.