应用套式PCR提高肺炎支原体P1-RFLP基因分型检测敏感性  

Application of nested PCR in P1-RFLP for genotyping Mycoplasma pneumoniae strains

作　　者：薛冠华[1] 王丽琼[1] 闫超[1] 冯燕玲[1] 赵汉青[1] 李少丽[1] 孙红妹[1] 

机构地区：[1]首都儿科研究所细菌研究室,北京100020

出　　处：《中华微生物学和免疫学杂志》2014年第2期141-145,共5页Chinese Journal of Microbiology and Immunology

基　　金：北京市自然科学基金资助（7112019）

摘　　要：目的探索一种适用于临床标本检测的肺炎支原体套式PCR．P1一RFLP基因分型方法并对其应用进行评价。方法参考肺炎支原体标准株P1基因RepMp4及RepMp2／3序列，分别在两个序列内部设计4条内套引物，采用文献报道的外套引物及新设计内套引物进行套式PCR扩增，扩增产物混合后用HaeⅢ酶切，根据不同的酶切图谱结果判断其型别，同时采用测序验证；对标准株DNA及临床标本DNA倍比稀释后进行检测，验证新改良方法敏感性；收集2012年肺炎支原体感染阳性标本，采用研究新改良的方法进行分型检测并与现有方法进行比较。结果经测序验证，以套式PCR为基础的P1-RFLP基因分型方法能够对临床标本进行很好的扩增；与现有的分型方法相比，新改良方法敏感性提高10^3倍，差异有统计学意义；北京地区2012年儿科肺炎支原体流行基因型为I型。结论套式PCR—P1-RFLP基因分型方法敏感性高，能够对临床标本进行很好的扩增。Objective To evaluate the efficiency of using nested PCR in restriction fragment length polymorphism analysis （P1-RFLP） for genotyping Mycoplasma pneumonia （M. pneumonia） in clini- cal specimens. Methods Based on the gene sequence of RepMp4 and RepMp2/3 in P1 gene of reference strains M129 （type 1 ） and FH （type 2） , two sets of inner primers were designed with a Haem restriction enzyme site （GGCC）. The nested PCR was set up to detect the target DNA in clinical specimens. The am- plification products were mixed and digested with Hae Ⅲ enzyme. The genotypes were analyzed by comparing with various restriction maps and the results were verified by sequencing analysis. The concentration of DNA extracted from standard and clinical strains were detected by ten-fold dilution to evaluate the sensitivity of nested PCR-PI-RFLP and P1-RFLP. M. pneumonia-positive specimens isolated from Beijing in 2012 were analyzed by the nested PCR-P1-RFLP and the results were compared with those by P1-RFLP analysis. Re- suits The nested PCR-P1-RFLP could effectively genotype M. pneumonia in clinical specimens and the re- sults were consistent with those by sequencing analysis. The sensitivity of new assay was 103 times higher than that of the original P1-RFLP. Of the 115 M. pneumoniae positive clinical specimens, 97.4% （ 112/ 115） were type 1 and the rest were type 2. Conclusion The nested PCR-P1-RFLP shows high efficiency for genotyping of M. pneumonia in clinical specimens. It might be useful for the surveillance of M. pneumoni- ae infection.

关 键 词：肺炎支原体 基因分型 套式PCR P1-限制性片段酶切长度分析 

分 类 号：R440[医药卫生—诊断学]