两种甲病毒基因组序列的一步RT-PCR检测

Detection of the genomic sequences of two alphaviruses by one-step RT-PCR

目的 通过对不同扩增条件的优化 ,建立两种马脑炎病毒的快速RT -PCR检测方法。方法 根据东部马脑炎病毒和西部马脑炎病毒基因组相应序列设计引物 ,然后采用两步法及两种一步法分别对其基因组序列进行RT -PCR扩增 ,并用琼脂糖凝胶电泳进行观察。结果与结论 三种方法均可从两种马脑炎病毒感染的乳鼠脑和细胞上清中扩增出单一的DNA片段 ,其大小与预期的相一致。与其他两种方法相比 ,本研究所建立的一步法更为简便快速、价格低廉 ,为进一步组装这些病毒的检测试剂盒奠定了基础。

Aim To establish a rapid RT PCR method for detection of two equine encephalitis viruses by optimizing reaction conditions Methods Primers used for EEE and WEE were designed basing on the sequences of their genomes Their genomic sequences were amplified by using two step RT PCR and two kinds of one step RT PCR The amplified products were analyzed routinely on agarose gels containing ethidium bromide Result and Conclusions A single DNA fragments of two viruses were amplified from the infected brains of suckling mice and culture cell supernatants by three RT PCR assays The sizes of the amplified fragments were equal to those of the expected products The one step RT PCR assay established in this study was more simple,time saving and cheaper in comparison with another two assays This study can lay a foundation for developing detection kits for these viruses