柯萨奇病毒B5巢式RT-PCR检测方法的建立

摘要

目的:建立一种快速、灵敏、特异的柯萨奇病毒B5(CVB5)检测方法.方法:根据GenBank 发表的CVB5河南分离株全基因组序列,在其VP1 区设计并合成巢式RT-PCR引物,优化PCR反应条件,建立检测CVB5的巢式RT-PCR方法.将已测得TCID50的病毒标准株进行10倍梯度稀释后用上述方法检测,以评价该方法的灵敏性;将5株不同的CVB5毒株和EV71等4株其他肠道病毒毒株及阴性对照用该方法进行扩增,以检测其特异性.用建立的巢式RT-PCR方法对52份病毒性脑炎病例样本进行检测.结果:该方法对CVB5的两轮PCR扩增敏感性分别为10 TCID50和10-4 TCID50;所有CVB5毒株用该方法扩增结果均为阳性,所选其他肠道病毒扩增结果均为阴性.52份临床病例样本中检出10份CVB5阳性,阳性率为19.23%.结论:所建立的CVB5巢式RT-PCR检测方法具有良好的灵敏度和特异度.%Aim:To establish a rapid , sensitive, and specific method to detect coxsackie virus B 5 ( CVB5 ).Methods: Two pairs of primers were designed according to the VP 1 section of CVB5 isolated from Henan province nucleoprotein gene sequences published in GenBank .With optimized reaction conditions , a reverse transcription nested PCR assay for detection of CVB5 was developed specifically.The standard virus strains which had been identified as TCID50 were serially tenfold diluted and were used to determine the sensitivity of the method , and cDNA extracted from five different CVB 5 strains and four other enteroviruses was used as template to assess the specificity of the method .Furthermore ,52 samples of viral encephalitis were performed by using this nested RT-PCR method.Results: The sensitivity of two-wheeled amplification of CVB5 were respectively 10 TCID50 and 10 TCID50.The amplification results of all CVB5 strains were positive , while the results of four other enterovimses were negative.Ten positive results were obtained from the 52 samples using this method and the positive rate was 19.23%.Conclusion: A nested RT-PCR assay to detect CVB5 has been established successfully.