应用whaF基因序列鉴定20型肺炎链球菌血清型

Identification of Streptococcus pneumoniae serotype 20with wha F gene sequencing

目的应用wha F基因测序方法,对20型肺炎链球菌进行血清型鉴定。方法采用血清凝集法和用20型肺炎链球菌型特异性基因(wci L基因)合成的引物进行PCR扩增,对中国医学细菌保藏管理中心所保藏的20型肺炎链球菌进行血清学鉴定;根据Gen Bank上发表的wha F基因设计合成引物,PCR扩增wha F基因,利用测序获得基因序列,分析wha F基因多态性。结果 7株20型肺炎链球菌菌株均能与20型血清产生血清凝集反应,wci L基因PCR扩增产物,可见与预期相符大小500 bp的特异性条带。wha F基因PCR扩增产物,除20-3外,其余6株菌均可见1 000bp大小与预期相符的PCR扩增产物。wha F基因测序和分析显示:20-2、20-4、20-5、20-7与20B亚型(Gen Bank accession No.:CR931679和JQ653093)的wha F基因序列相同;20-6与20A亚型(Gen Bank accession No.:JQ653094)的wha F基因序列相同;20-1与CR931679和JQ653093相比,在898有点突变(A→C)。20-3的wha F基因比20A亚型和20B亚型的wha F基因均少了约600 bp。结论 20-6为20A亚型肺炎链球菌,20-2、20-4、20-5、20-7均为20B亚型肺炎链球菌。

Objective To determine the subtypes of Streptococcus pneumoniae serotype 20 by using of wha F gene sequencing. Methods The serum agglutination and PCR amplification were carried out,later by using the primers designed for wci L gene specific for Streptococcus pneumoniae Serotype 20 in determination of the subtype of Streptococcus pneumoniae serotype 20 obtained from National Center for Medical Culture Collections. The primers were designed and synthesized according to the sequences of wha F gene published in the Gen Bank. The wha F gene was amplified by PCR amplification then followed by gene sequencing,and the gene polymorphism was analyzed. Results All 7 strains of Streptococcus pneumoniae serotype20 occurred positive results by agglutination with the serotype 20 antisera. The PCR amplification product of wci L gene could be observed with an expected specific band about 500 bp,and wha F gene with an expected specific band about 1 000 bp in all strains except for strain 20-3. The sequencing and analysis of wha F gene showed that the sequences of strain 20-2,20-4,20-5 and 20-7 were identical with that of subtype 20B（ Gen Bank accession No. ： CR931679 and JQ653093）; the sequence of strain 20-6 was identical with that of subtype 20A（ Gen Bank accession No. ： JQ653094）. Compared to CR931679 and JQ653093,the sequence of strain 20-1 had a point mutation in position 898（ A→C）. The sequence of wha F gene in strain20-3 was shortened by about 600 bp compared to that of either subtype 20 A or 20 B. Conclusion Strain 20-6 was Streptococcus pneumoniae subtype 20 A,but strain 20-2,20-4,20-5 and 20-7 were Streptococcus pneumoniae subtype 20 B.