应用PCR技术和wciP基因测序方法鉴定6群肺炎链球菌血清型

PCR analysis and wciP gene sequencing for determining the serotypes of Streptococcus pneumonia se-rogroup 6 strains

目的：利用分子生物学方法，包括PCR技术和wciP基因测序鉴别6群肺炎链球菌血清型。方法根据GenBank上发表的wchA基因和wciO基因设计合成6群特异性引物，wciNα基因设计合成6A/B型特异性引物，wciNβ基因设计合成6C/D型特异性引物，做PCR扩增，凝胶电泳确认PCR产物；根据wciP基因设计6群特异性引物，PCR扩增后产物测序确定第584位核苷酸。结果原6A型9株菌株（编号为6A-1至6A-9）和原6B型7株菌株（编号为6B-1至6B-7）用6群特异性引物进行的扩增均有分子量在2.0 kb左右的PCR产物，而1型没有PCR产物；除6A-2、6A-3、6B-3和6B-7外，其余菌株用6A/B型特异性引物进行的扩增有600 bp左右的PCR产物；6A-2、6A-3、6B-3和6B-7用6C/D型特异性引物进行的扩增有750 bp左右的PCR产物；测序结果确认原6A型9株菌株wciP基因第584位为G，原6B型7株菌株wciP基因第584位为A。结论6A-1、6A-4、6A-5、6A-6、6A-7、6A-8、6A-9属于6A型，6A-2、6A-3属于6C型，6B-1、6B-2、6B-4、6B-5、6B-6属于6B型，6B-3和6B-7属于6D型。

Objective To identify the serotypes of Streptococcus pneumonia serogroup 6 strains by using polymerase chain reaction （ PCR ） analysis and wciP gene sequencing .Methods Specific primers were designed based on the gene sequences of wchA gene,wciO gene,wciNαgene,wciNβgene and wciP gene published in GenBank ,and used for PCR analysis .PCR products of wciP gene were sequenced .Results The PCR products of all 16 Streptococcus pneumonia serogroup 6 strains （6A-1 to 6A-9,6B-1 to 6B-7） were about 2.0 kb by using the serogroup 6 specific primers .PCR products of about 600 bp were amplified from the strains except for 6A-2,6A-3,6B-3 and 6B-7 by using the 6A/B specific primers.The strains of 6A-2,6A-3, 6B-3 and 6B-7 had about 750 bp PCR products by using primers specific for 6C/D.The sequencing analysis showed that the wciP gene at 564 nt site were G for all 6A strains,and A for 6B strains.Conclusion Using PCR assay,16 strains of serogroup 6 Streptococcus pneumonia were typed to four serotypes including serotype 6A （6A-1,6A-4,6A-5,6A-6,6A-7,6A-8 and 6A-9）,serotype 6C （6A-2 and 6A-3）,serotype 6B （6B-1, 6A-2,6B-4,6B-5 and 6B-6） and serotype 6D （6B-3 and 6B-7）.