RT-PCR检测番茄不孕病毒

Detection of Tomato aspermy virus by RT-PCR.

根据番茄不孕病毒 (TAV)RNA3上外壳蛋白基因序列设计、合成引物 ,以感病组织和健康组织总RNA为模板 ,进行cDNA合成和PCR扩增 ,结果从感病组织中扩增出与预期的6 42bp大小一致的目标片段 ,而健康组织无此扩增产物 ;将PCR产物插入 pGEM T载体克隆并测序 ,序列分析表明与TAV不同株系的序列同源性达到 96 2 %以上 ;将感病组织总RNA以 1 0倍梯度稀释成不同浓度 ,测出RT PCR检测TAV的灵敏度为 1 0 -5 ;PCR产物克隆作为RT -PCR反应的阳性对照 ,解决了毒源保存和传播的问题 ,从而建立了TAV快速、灵敏、准确的RT

A pair of primers were designed and synthesized based on the nucleotide sequence of coat protein gene of TAV3 from England.The expected size 642bp was amplified by RT-PCR method from the infected sample,but not from health sample.The amplified products were cloned into pGEM T easy vector and sequenced.The nucleotide sequence was compared with different strains of TAV.The result showed that their homology attained up 96 2%.The total RNA from infected sample was diluted to a series of 1∶10～1∶10 -6 and the detection sensitivity of RT-PCR was 10 -5 .It has resolved the problem of storing and preventing spread of virus source that the clone of PCR product regard as positive control of PCR.So the rapid,sensitive and accurate identification and detection method by RT-PCR is built.