肺炎支原体多位点SNP基因分型方法的建立与应用

Establishment of a new technique for genotyping multiple single nucleotide polymorphisms in Mycoplas-ma pneumoniae

目的基因分型技术是研究肺炎支原体(MP)抗原变异和流行暴发的重要手段,本研究拟建立一种适用于我国MP菌株的新型多位点SNP基因分型技术(以下称新SNP分型法)。方法对69株MP全基因组序列使用SNP stats软件获取全部SNP位点。使用连续体外传代10代的18株MP菌株对初筛的34个SNP位点进行稳定性验证,使用40株MP临床株进行SNP位点区分能力验证,建立多位点SNP分型方法。选取160株MP国内菌株进行SNP分型、传统两种基因分型、MLVA分型以及现有4种SNP分型方法的比较。结果最终选取的6个SNP位点组合可将MP分成SNP-00~SNP-64共65种基因型,其中SNP奇数型对应传统type1基因型MP,SNP偶数型对应传统type2基因型MP。160株国内MP菌株经本研究建立的SNP分型方法分为14个基因型,其分型结果与type1和type 2两种基因型一致性为100%(160/160)。MLVA方法分为4种基因型,传统SNP分型方法(以下称SNP分型法)分为3种基因型。其中35例聚集性病例分离株经新SNP分型法分为SNP-03和SNP-15两种基因型,其余3种方法无法区分。结论本研究建立的并报SNP分型方法能兼容传统两种基因型分型,并且增加了基因型区分能力,对国内MP的分型能力优于现有的多种基因分型技术,可用于我国MP的抗原变迁及区域性流行预警监测以及局部MP爆发的溯源研究。

Objective Genotyping is a crucial technique for monitoring antigenic variation and outbreaks of Mycoplasmapneumoniae.The aim of this report was to establish a novel technique suitable for genotyping multiple single nucleo-tide polymorphisms(SNPs)in M.pneumoniae strains in China.Methods The software SNP stats was used to obtain all SNP loci in the entire genomic sequence of 69strains of M.pneumoniae.The stability of the 34SNP loci with primary screening was verified using 18 M.pneumoniae strains with 10passages in vitro.Forty clinical M.pneumoniae strains were used to verify the discrimination power of SNP loci.One hundred and sixty domestic strains of M.pneumoniae were selected for genotyping,and results of using this SNP method,p1gene-based typing,the MLVA method,and a published SNP method were compared.Results Based on a combination of six SNP loci,M.pneumoniae was grouped into 65genotypes.The odd SNP type corresponds to the traditional genotype 1of M.pneumoniae,and the even SNP type corresponds to the traditional genotype 2of M.pneumoniae.In this study,160 M.pneumoniae strains from China were grouped into 14genotypes using SNP typing;those genotypes were identified as genotype 1and genotype 2.The MLVA method identified 4genotypes,and the current SNP typing identified 3genotypes.Only SNP genotyping was able to distinguish the 2genotypes SNP-03and SNP-15in 35clustered cases;the remaining three methods were unable to do so.Conclusion SNP typing is not only compatible with the traditional 2genotypes but it also has increased ability to differentiate genotypes,and this method has superior ability to type M.pneumoniae in China.This method may play an important role in monitoring antigenic changes in M.pneumoniae in China,providing early warnings of regional epidemics,and facilitating the traceability of local M.pneumoniae outbreaks.