利用RT-PCR方法检测甜瓜种子中南瓜花叶病毒

Detection of Squash mosaic virus from muskmelon seeds by RT-PCR

通过设计特异性引物SQCPF/SQCPR,扩增南瓜花叶病毒(Squash mosaic virus,SqMV)CP基因的保守序列,进行RT-PCR,对扩增得到的特异性片段进行序列测定,测序结果显示扩增产物序列与GenBank中登录的SqMV外壳蛋白基因存在87%～96%的一致性;建立了适用于检测甜瓜种子中南瓜花叶病毒的RT-PCR结合ELISA方法,通过在酶联板的反应孔中直接进行反转录合成cDNA,能扩增到预期大小的DNA条带,且检测灵敏度高于DAS-ELISA方法10倍以上。用建立的RT-PCR结合ELISA方法检测进境甜瓜种子携带的SqMV,在42份样品中检出4份阳性样品。

RT-PCR and ELISA were set up for detection of Squash mosaic virus （SqMV） from muskmelon seeds. The primers of SqMV were designed on the basis of coat protein genes of SqMV. Products of SqMV by RT-PCR was sequenced. The results showed that the sequence shared 87% --96% nucleotide sequence identity with SqMV coat protein genes. RT-PCR integrated with ELISA revealed more than 10 times higher sensitivity than ELISA alone. Four positive samples were detected among 42 melon seed lots by using RT-PCR integrated with ELISA method.