多重PCR法用于脑膜炎奈瑟菌血清群分群鉴定的适用性  

Applicability of multiplex PCR for serogrouping of Neisseria meningitidis

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作  者:张丽芝 白贵杰 杨英英 任克明 刘方蕾 罗树权 沈荣 ZHANG Li-zhi;BAI Gui-jie;YANG Ying-ying;REN Ke-ming;LIU Fang-lei;LUO Shu-quan;SHEN Rong(First Department of Research,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区:[1]兰州生物制品研究所有限责任公司第一研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出  处:《中国生物制品学杂志》2020年第12期1414-1420,共7页Chinese Journal of Biologicals

基  金:兰州市2018年第四批科技计划项目十大科技创新项目(2018-4-14)。

摘  要:目的应用多重PCR法鉴定脑膜炎奈瑟菌(Neisseria meningitidis,Nm)A、B、C、W135、Y 5个血清群,验证在基因水平鉴定疫苗用菌种血清群的适用性,并与实验室经典细菌培养法和血清凝集试验进行比对。方法提取标准Nm血清群A、B、C、W135、Y的基因组DNA,针对5个血清群Nm荚膜多糖合成基因上的特异序列,应用多重PCR方法对目的片段进行扩增,根据PCR扩增产物的碱基片段大小不同区分这5个血清群;采用PCR方法鉴定7株Nm分离菌种的血清群;并与实验室传统细菌培养法和血清玻片凝集试验比对。结果多重PCR方法能够鉴定Nm,并同时完成血清分群鉴定。该方法特异性和灵敏度(最低检测限为基因组DNA 0.1 ng)均较高。7株Nm分离菌种PCR检测结果与实验室经典培养法和血清玻片凝集试验结果一致,其中1株为血清群A群,1株B群,2株C群,1株W135群,2株Y群。结论多重PCR方法对Nm检测的特异性和灵敏度均优于传统细菌培养法和血清玻片凝集试验,可同时检测Nm菌种的种特异基因和群特异基因,提高了分群鉴定的准确性。此外,试验结果客观,且可长期保存和追溯性查询,有利于疫苗用菌种的质量控制。Objective To identify five serogroups of Neisseria meningitides(Nm),A,B,C,W135 and Y,by multiplex PCR,verify the applicability of the method for identification of serogroups of bacterial strains for vaccines at gene level and compare the result with those by classical laboratory bacterial culture and serum agglutination test.Methods The genomic DNAs of standard Nm strains of serogroups A,B,C,W135 and Y were extracted,based on which the target gene fragments were amplified by multiplex PCR according to the specific sequence of synthetic capsular polysaccharide genes of five serogroups.The serogroups of Nm strains were distinguished according to the size of base fragments of PCR products.The serogroups of seven Nm isolates were identified by PCR,and the results were compared with those by classical laboratory bacterial culture and serum agglutination test.Results Multiplex PCR was able to be used for the identification and serogrouping of Nm,which showed high specificity and reproducibility,with a minimum detection limit of 0.1 ng of genomic DNA.All the test results of seven Nm isolates were consistent with those by classical laboratory bacterial culture and serum agglutination test.One of the seven strains was of serotype A,while one was of serotype B,two were of serotype C,one was of serotype W135,and two were of serotype Y.Conclusion The specificity and sensitivity of multiplex PCR for detection of Nm were higher than those of classical laboratory bacterial culture and serum agglutination test.The species-and serogroup-specific genes of Nm strains were detected at the same time,which improved the accuracy of serogrouping.In addition,the test results were objective,persistent and traceable,which was beneficial to the quality control of vaccine strains.

关 键 词:多重PCR 脑膜炎奈瑟菌 基因分群 

分 类 号:Q939.99[生物学—微生物学]

 

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