猴轮状病毒(SA11)RT-PCR检测方法的建立

Establishment of the Detection Meth od of Simian Rotavirus (SA11)Using R everse Transcriptase and Pol ymerase Chain Raction

根据具有高度保守性的编码VP7轮状病毒糖蛋白的基因9序列设计引物,分别进行RT-PCR和NT-PCR扩增,结果:引物Beg9/End9、Beg9-1/End9-1及引物RVG9/aET3、RVG9/aET3-1分别能在一次RT-PCR扩增中出现预期的1062bp和374bp扩增带;引物RVG9/aET3、RVG9/aET3-1在NT-PCR扩增中均能出现预期的374bp扩增带;验证了猴轮状病毒SA11属于血清型3;引物RVG9/aET3进行敏感试验能检测到0.5pg的轮状病毒(SA11)dscDNA。

The simian virus(SA11)gene segment coding the major outer c apid glycopr otein vp7was amplified directly from SA11by reverse transcriptase and polymer a se chain reaction(RT-PCR).Two dscDNA fragment of the expected size(10 62bp and 374bp)was ampliflied from simian virus(SA11)by(RT-PCR)and a dscDNA fragment of the expected size(374bp)was ampliflied by NT-PCR,b ut no band was observed in other control samples.The simian virus(SA11)were assigned the serotype3by PCR using the serotype-specific primers.The sen si tivity of the reaction was determi ned with different concentrations o f dscDNA .As littler as 0.5pg dscDNA could be detected.