基于外壳蛋白基因序列的百香果CMV快速检测

Rapid detection of passion fruit CMV based on coat protein gene sequence

为提高百香果黄瓜花叶病毒(Cucumber mosaic virus,CMV)的检测效率,根据Gen Bank数据库中的CMV外壳蛋白(coat protein,CP)的基因核苷酸序列保守区域设计引物,在单一PCR检测体系的基础上,建立双重PCR检测体系.结果发现:样本中含有2个CMV的外壳蛋白基因序列,分别命名为PeCMV-LJ和PeCMV-1.测序结果显示,克隆的PeCMV-LJ和PeCMV-1重合片段只相差一个碱基,基因序列大小分别为619、301 bp;生物信息学分析显示,克隆的PeCMV-LJ和PeCMV-1片段与已报道的其他CMV分离物的一致性介于94.84%~97.58%;CMV的CP基因进化分析结果表明,百香果CMV与黄瓜CMV进化关系最近.利用3条百香果CMV的CP基因特异引物的设计,可同时检测病毒3个特异性位点,避免假阳性的产生,1对百香果EF1a内参引物的使用,可以检测百香果cDNA合成质量,避免假阴性的产生,整个检测过程可以在4 h内完成.综上,本研究建立的双重PCR体系能够用于CMV的快速检测.

To improve the detection efficiency of Cucumber mosaic virus(CMV),primers have been designed based on the conservative region of the CMV coat protein(CP)gene nucleotide sequence in the Gen Bank database,and a dual PCR detection system based on a single PCR detection system as well.The result showed that:the sample contained CMV coat protein gene sequences,named PeCMV-LJ and PeCMV-1.The sequencing results showed that the cloned PeCMV-LJ and PeCMV-1 conincident fragments differed by only one base,and the gene sequences were 619 bp and 301 bp,respectively.Bioinformatics analysis showed that the consistency of the cloned PeCMV-LJ and PeCMV-1 fragments with other reported isolates was 94.84%-97.58%.The results of CMV CP gene evolution analysis showed that the evolutionary relationship between the host Passion fruit and cucumber was the closest,3 specific primers were designed accord to the Passion fruit cucumber mosaic virus isolates coat protein genes,those primes can be using in detectiing 3 specific sites of the virus in one reaction to avoid the generation of false-positive results.The EF1a reference primes of the passion fruit can be using in inspecting the cDNA synthesis quanlity to avoid the generation of false-negative results.The whole virus-free inspection process can be completed in 4 hours.The established dual PCR system could be used for the rapid detection of CMV.