猴空泡病毒40核酸序列检测法的优化研究

摘要

Objective To optimize the PCR primer sets for Simian virus 40 (SV40) detection and establish an assay method for SV40 which is of high sensitivity, strong specificity, broad applicability. Methods Two pairs of PCR primers were designed of based on 21 different SV40 strains genome by Primer Premier 5.00 software, and the features of two pairs of PCR primers were analyzed by Oligo software (version 6.71), conservative nucleotide of two pairs of PCR primers and the PCR amplification product were analyzed by DNAMAN software (version 6.0.40). Two pairs of new-built PCR primers were compared with those derived from China pharmacopoeia (Clip) in these aspects. The detection sensitivity of four pairs of PCR primers were analyzed using different SV40 DNA diluent as PCR template. The detection specificity of four pairs of PCR primers were analyzed using sterile water, Vero cell DNA, SV40 DNA as PCR template, respectively. Results The sequences of the new PCR primer sets VP1 and T are conservative for 21 Strains. The sequences of PCR primer sets GCVP1 and GCT are conservative for SV40 strains whose accession No. is J02400, NC_001669, AF316139 and AF316141. As far as the same diluent SV40 DNA template is concerned, the PCR amplification efficiency of PCR primer set VP1 and T is higher than that of PCR primer set GCVP1 and GCT. There are non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets GCVP1 and GCT, whereas there are no non-specific band in nucleic acid electrophoresis for amplification products of PCR primer sets VP1 and T. Conclusion The new assay method for SV40 nucleic acid sequence has many better qualities than those in Chp such as high sensitivity, strong specificity, broad applicability, conservation of primers and their amplification products and so on.%目的 对通常使用的猴空泡病毒40(Simian vacuolating virus 40,SV40)核酸序列检测法进行优化,寻找敏感性高、特异性强、适用面广的SV40核酸序列检测引物.方法 以21个SV40毒株完全基因组为基础数据,用Primer Premier 5.00软件重新设计两对SV40 DNA检测引物,用Oligo 6.71软件和DNAMAN 6.0.40软件对引物参数进行分析,将分析结果与通常使用的检测引物进行比较.用不同稀释度SV40核酸序列作模板,比较4对引物检测的敏感性.分别用无菌水、Vero细胞DNA、SV40 DNA作模板检测4对引物的特异性.结果 对于21个SV40病毒株,优化引物对VP1和T的序列是保守的;对于接受号为J02400、NC_001669、AF316139和AF316141的4个病毒株,通常使用的引物对GCVP1和GCT的序列是保守的;用同一稀释度的SV40 DNA作模板,引物对VP1和T的扩增效率明显高于引物对GCVP1和GCT;在特异性检测比较中,引物对VP1和T没有出现非特异性扩增条带,引物对GCVP1和GCT在100 bp处出现非特异性扩增条带.结论 优化的SV40核酸序列检测法具有敏感性高、特异性强、检测面广、引物及其PCR产物序列保守等特点.