半巢式多重PCR法对A组轮状病毒VP7基因的分型

Group A rotavirus VP7 genotyping by semi-nested multiplex-PCR

目的采用半巢式多重PCR法对A组轮状病毒(group A rotavirus,RVA)VP7基因进行分型。方法根据GenBank中RVA VP7基因5′端保守区设计合成多组基因分型引物,提取11个RVA代表流行毒株病毒的RNA,采用半巢式多重PCR方法进行RVA的VP7基因分型,使用BLAST在线软件与GenBank上相应VP7核苷酸序列比对。结果经琼脂糖凝胶电泳鉴定,11株RVA的多重PCR产物大小均与预期一致,能正确鉴别具有代表性的流行毒株VP7基因型[G1、G2、KN105(Wa-like G3)、To16-01(equine-like,DS-1-likeG3)、G4、G8、G9及G12];经BLAST在线软件比对,RVA病毒株VP7基因序列与设计的上游引物匹配,并与下游引物互补,引物特异性良好。结论利用设计的引物成功建立的半巢式多重PCR方法能正确识别RVA的VP7基因型。

Objective To analyze the genotype of group A rotavirus(RVA) VP7 gene by semi-nested multiplex PCR.Methods Multi-component primers were designed according to the sequence of conserved region at 5′-terminus of VP7 gene of RVA in GenBank,with which the RNAs of 11 representative RVA strains were extracted for VP7 genotyping by semi-nested multiplex PCR. The result was compared with the corresponding VP7 nucleotide sequences in GenBank by using BLAST online software. Results Agarose gel electrophoresis showed that all the lengths of PCR products of 11 RVA strains were consistent with those expected. The genotypes of VP7 gene of representative RVA strains,such as G1,G2,KN105(Wa-like G3),To16-01(equine-like,DS-1-like G3),G4,G8,G9 and G12 were identified correctly. The comparison by using BLAST online software showed that the VP7 gene sequence was matched to the designed upstream primers and complementary to the downstream primers,indicating good specificity of the primers. Conclusion The developed semi-nested multiplex PCR method by using the designed primers may be used for the correct recognition of VP7 genotype of RVA.