摘要
目的明确19群肺炎链球菌(Streptococcus pneumoniae)国家标准菌株的分子特征,为完善中国肺炎链球菌国家标准菌株的质量标准提供依据。方法在传统肺炎链球菌菌种检定方法的基础上,应用16S rRNA基因分析、聚合酶链式反应(polymerase chain reaction,PCR)血清分型、多位点序列分型(multilocus sequence type,MLST)和脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型等多种分子生物学质控方法,对来源于中国医学细菌保藏管理中心的20株19群肺炎链球菌国家标准菌株进行分析。结果20株19群肺炎链球菌的16S rRNA基因序列与肺炎链球菌模式株NCTC 7465的16S rRNA基因序列的相似性在99.64%~100%之间,碱基差异0~5 bp。PCR血清分型结果表明:11株19F型菌株均检测到大小为304 bp的19F型特异性扩增条带,6株19A型菌株均检测到大小为478 bp的19A型特异性扩增条带,PCR血清分型结果与血清凝集试验结果一致。MLST分型结果表明,相同血清型的菌株可以具有不同的序列型(sequence type,ST)。共获得12个ST型,其中6个ST型(10496、10497、10501、10502、10503和10509)为首次报道。PFGE分型结果显示:各型别菌株各具有其特征性PFGE带型(9~17条带),相同血清型或ST型菌株可能具有不同的PFGE带型。共存在18种不同的PFGE带型,其中19F型和19A型菌株中各有一对菌株的ST型和PFGE图谱完全一致。菌株的传代稳定性考察结果显示:在25代以内31708菌株的PFGE带型未发生变化,遗传特征是稳定的。结论16S rRNA基因分析、PCR血清分型、MLST分型和PFGE分型等分子生物学方法应用于中国肺炎链球菌国家标准菌株的质量控制,可获得更加全面的肺炎链球菌标准菌株身份信息数据(包括序列、PCR扩增片段、序列型、图谱等),为进一步完善中国肺炎链球菌国家标准菌株的质量标准提供依据和数据支撑。
Objective The aim of the study is to clarify the molecular characteristics of the national standard strains of serogroup 19 Streptococcus pneumoniae,which could provide a basis for improving the quality standards in relation to this strain in China.Methods Besides the traditional quality control methods used for Streptococcus pneumoniae,various molecular quality control methods,including 16S rRNA gene analysis,PCR serotyping,multilocus sequence type(MLST)and pulsed field gel electrophoresis(PFGE),were applied to analyze 20 national standard strains of serogroup 19 Streptococcus pneumoniae from National Center for Medical Culture Collections(CMCC).Results The 16S rRNA gene similarity of 20 strains of serogroup 19 Streptococcus pneumoniae were 99.64%to 100%compared to that of type strain NCTC 7465,and the base difference was between 0 and 5 bp.The PCR-based serotyping showed that the 304 bp PCR products were detected in all the 11 strains of 19F,the 478 bp PCR products were detected in all the 6 strains of 19A,and PCR-based serotyping was consistent with serum agglutination test.The MLST typing indicated that strains with the same serotype could have different sequence types(ST).A total of 12 STs were obtained,among which 6 ST types(10496,10497,10501,10502,10503 and 10509)were firstly reported.The PFGE showed that all types of strains had their characteristic PFGE patterns(9 to 17 bands),and the strains with same serotype or ST type may have different PFGE patterns.A total of 18 different PFGE patterns were detected,among which one pair of the 19F or 19A strains showed the identical ST and PFGE patterns.The PFGE patterns of 31708 strain did not change and the genetic characteristics were in a stable state within 25 generations.Conclusion It is by using molecular quality control methods,including 16S rRNA gene analysis,PCR serotyping,MLST typing and PFGE resulted in a resonable supportive evidence,that a more comprehensive identity information for national standard strains of Streptococcus pneumoniae will be further improved in China.
作者
李康
黄洋
徐潇
陈琼
王春娥
石继春
陈翠萍
叶强
LI Kang;HUANG Yang;XU Xiao;CHEN Qiong;WANG Chun-e;SHI Ji-chun;CHEN Cui-ping;YE Qiang(Division of Respiratory Bacterial Vaccines,Institute for Biological Product Control,National Institutes for Food and Drug Control,National Center for Medical Culture Collections,Beijing 102629,China)
出处
《微生物学免疫学进展》
2019年第6期20-27,共8页
Progress In Microbiology and Immunology
基金
国家科技重大专项经费资助(2018ZX09738006-006)
关键词
19群肺炎链球菌
聚合酶链式反应血清分型
多位点序列分型
脉冲场凝胶电泳
质量控制
Serogroup 19 Streptococcus pneumoniae
PCR-based serotyping
Multilocus sequence type(MLST)
Pulsed field gel electrophoresis(PFGE)
Quality control
作者简介
李康(1982-),男,博士,主要从事呼吸道细菌疫苗质量控制及国家标准医学菌种检定和研究工作;通信作者:叶强,主任技师,E-mail:qiangyee@nifdc.org.cn。
