利用Real-time RT-PCR方法检测苹果中苹果茎沟病毒

Quantitative detection of Apple stem grooving virus from apple by Real-time RT-PCR

以含有苹果茎沟病毒(Apple stem grooving virus,ASGV)的苹果枝条为试材,设计扩增产物片段为176bp的特异性引物,优化反应条件,构建标准曲线,建立了苹果茎沟病毒的SYBR Green I实时荧光定量RT-PCR检测方法。对该方法进行了灵敏度和重复性试验,并且利用建立的方法对受ASGV感染的苹果枝条和树叶进行定量检测。最后,比较了实时荧光定量RT-PCR与常规RT-PCR检测的灵敏度。结果表明:样品的荧光定量PCR有良好的扩增曲线和溶解曲线,该方法的灵敏度可达6.8×102拷贝/μL,检测其灵敏度是传统RT-PCR的100倍,所建立的荧光定量技术可用于田间样品中此病毒的检测,丰富了ASGV的检测手段,提高了检测灵敏度,具有较好的应用前景。

A quantitative and SYBR Green I based Real-time PCR was developed to detect Apple stem grooving vi- rus （ASGV） using branches of apple. Sensitivity and reproducibility of this method were verified. In this study, a pair of specific primers of Apple stem grooving virus was designed, with an expected PCR product of 176 bp in length. Then the method was developed by constructing a control curve and optimizing the reaction conditions. The Results showed that the Real-time PCR had a good amplification curve and dissolve curve, and the sensitivity of this method was proved to be 6.8×10^2 copies/μL, which was about 100 times higher than that of the conven- tional RT-PCR assay. The Real-time RT-PCR described here is easy to operate, and also improve the detection sensibility and enrich the detection methods of ASGV. The detection system established in this study will be useful for detection of ASGV from infected field samples.