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目的设计A组轮状病毒RV特异性引物,应用Tth酶,建立一步法RT-PCR扩增方案,检测西安地区腹泻幼儿196份粪便标本,并与本室研制建立的反向间接血凝法RPHA,聚丙烯酰胺凝胶电泳,市售ELISA试剂盒平行检测对比分析。方法设计并合成第九基因序列保守区互补的特异性引物,在经典法RT-PCR试验成功的基础上,建立合理的一步法RT-PCR。结果四种检测结果阳性率分别为33.16%,23.47%,21.94%和25%,X2检验表明,RT-PCR最为敏感。结论反转录和PCR由Tth酶一步完成,克服了经典法中AMV、RNasin等试剂昂贵,操作繁锁等缺点,很适合临检需要。
Objective To design a group A rotavirus RV-specific primer and use Tth enzyme to establish a one-step RT-PCR amplification program to detect 196 stool specimens of children with diarrhea in Xi’an area and to establish reverse indirect hemagglutination assay , Polyacrylamide gel electrophoresis, commercial ELISA kit parallel detection comparative analysis. Methods Specific primers complementary to the conserved region of the ninth gene were designed and synthesized. Based on the successful RT-PCR experiments, a reasonable one-step RT-PCR method was established. Results The positive rates of the four tests were 33.16%, 23.47%, 21.94% and 25%, respectively. The results of X2 test showed that RT-PCR was the most sensitive. Conclusion Reverse transcription and PCR were completed in one step by Tth enzyme, which overcomes the shortcomings of classic reagents such as AMV and RNasin which are expensive and cumbersome to operate. It is suitable for clinical examination.