黄瓜绿斑驳花叶病毒实时荧光定量PCR检测体系的建立

Detection of cucumber green mottle mosaic virus by using real-time fluorescence quantitative PCR

为灵敏、快速地检测西瓜种子和果实中的黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV),根据该病毒的衣壳蛋白(coat protein,CP)序列设计特异性引物,建立基于SYBR GreenⅠ染料的实时荧光定量PCR(real-time fluorescent quantitative PCR,RT-qPCR)技术,并对采集的12份不同发育期西瓜果实样品进行检测。结果表明,建立的RT-qPCR检测技术可对西瓜种子中的CGMMV含量进行精确检测,检测下限为3.35×10^(2) copies/μL。采集的12份不同发育期西瓜果实样品中有10份被检测出携带CGMMV,病毒含量范围为1.00×10^(4)~4.80×10^(6) copies/μL。随着果实发育,由CGMMV引起的果实倒瓤症状愈加明显,果实中CGMMV的积累量也明显增加。授粉后25 d西瓜果实中CGMMV的含量是授粉后10 d时的30倍,表明病毒在果实中的积累可能受生长发育期的影响且果实倒瓤症状的形成与病毒积累量呈正相关。

For the sensitive and rapid detection of cucumber green mottle mosaic virus(CGMMV)in watermelon seeds and fruits,a real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)method based on SYBR GreenⅠfluorescent dye was developed.Using this technique,12 watermelon fruit samples collected at different developmental stages were tested.The results showed that the method could be accurately and sensitively detect CGMMV in watermelon seeds.The detection sensitivity was above 3.35×10^(2) copies/μL.In total 12 watermelon fruit samples,ten samples were positive for CGMMV,with a content of 1.00×10^(4)-4.80×10^(6) copies/μL,which increased significantly with the development of the fruit,while the symptoms become more obvious.The virus content of CGMMV in watermelon fruits 25 days after pollination was about 30 times that of ten days after pollination,indicating that the accumulation of virus in the fruit might be related to the growth and development period of the watermelon fruit.The symptoms of fruit decay caused by CGMMV were highly correlated with virus accumulation.