多重PCR技术对贵州省肺炎链球菌脑膜炎病例病原分型的研究

Study on the multiplex PCR technology for determining serotypes of Streptococcus pneumonia meningitis case in Guizhou province

目的研究用多重PCR技术对肺炎链球菌脑膜炎的病原进行分型。方法获取患者脑脊液标本,离心,上清液用乳胶凝集试剂进行抗原检测,沉淀进行革兰氏染色并提取其DNA。根据全国流行肺炎链球菌主要的血清型设计和合成9对特异性引物,优化多重PCR条件并对病原体DNA进行扩增,通过电泳对肺炎链球菌血清型进行初分类,然后用普通PCR对可疑血清型进行再次确定,最后对PCR产物进行测序和序列分析。结果乳胶凝集试验出现肺炎链球菌乳胶凝集颗粒,多重PCR分别在约600bp和220bp处出现阳性条带,考虑为CPSA和6B血清型;普通PCR进一步确认为CPSA和6B。测序后分析为肺炎链球菌血清群6,血清型为6A/6B/6C/6D-f。结论多重PCR技术由于使用的标本量少,且可以提高诊断速度、经济,故可以直接用于临床标本的肺炎链球菌血清分群/型。

Objective Study the serotyping of the pathogen of Streptococcus pneumonia meningitis with multiplex PCR technology. Methods Got the cerebrospinal fluid of the case, centrifugated and tested the antigen of the supernatant by latex agglutination kit, and done Gram staining and extracted DNA of the precipitation, and designed and synthesized 9 pairs primer of main serotypes of S. pneumonia which were epidemic in the nation, and optimized PCR conditions, DNA amplification and electrophoresis, classified roughly with multiplex PCR technology, and then confirmed by normal PCR, finally sequenced for the PCR product and analyzed. Results Latex agglutination experiment showed positive in the S. pneumon/a. The multiplex PCR showed positive of serotype CPSA and 6B. The normal PCR showed the same result. Analyzing the sequence of PCR product, the pathogen of meningitis was S. pneumonia serogroup 6, and the serotype was 6A/6B/6C/6D-f. Conclusions Multiplex PCR technology could use in serogroups or serotypes of S. pneumonia clinical sample, because the technology need less samnle, and could increase diagnose speed, and was economic.