马铃薯Y病毒一步RT-PCR检测试剂盒的研制

One-step RT-PCR Kit Developed for Detection of Potato Virus Y

马铃薯Y病毒(Potato virus Y,PVY)对马铃薯的危害最大,可导致马铃薯退化,降低马铃薯产量。解决这一问题的重要途径就是培养脱毒种薯,但是否完全脱毒需要经过检测才能证实。本研究依据PVY CP基因序列设计合成了一对引物PY1、PY2,以带毒样品植物总RNA为模板,在同一个反应中同时加入反转录和PCR反应所需试剂,反应程序中包括反转录和PCR反应所需条件,进行反应扩增,带毒样品扩增得到340 bp的目的条带,而健康对照无此目的条带,从而建立了PVY的一步RT-PCR检测技术,并组装成试剂盒。该试剂盒具有良好的稳定性和特异性,灵敏度可以检测到带毒植物组织下限的6.25μg,高于ELISA(100μg)和NASH(15μg)的灵敏度,虽然和常规方法的灵敏度相同,但更为快速、简便、易于操作,适合脱毒苗和脱毒种薯生产单位做大量样品的检测。

Potato virus Y （PVY） is a main factor that affects the potato production, leading to significant yield losses and quality degradation. One of the ways to deal with this problem is to get virus-free plantlets by meristem tip culture, but whether or not the plantlets recovered from meristem culture is virus-free needs confirmation. Primers PY1 and PY2 were designed and synthesized based on PVY CP gene sequence. One fragment of 0.34 kb was amplified by one-step RT-PCR using the total RNA of the PVY infected plant; however, for the healthy sample nothing was got. One-step RT-PCR detection method for PVY is established by mixing all the reagents used in RT and PCR and designing the reaction conditions used in RT and PCR reactions based on the detection method for PVY. One-step RT-PCR detection kit accordingly was also established. The kit had high stability and specificity. The sensitivity of detection in leaves is about 100 μg by ELISA and 15 μg by NASH, while the sensitivity for the kit was about 6.25 μg. Though the sensitivity of the kit was the same as routine RTPCR, but the use of the kit was time-saving and cost-effective. So the kit can be used as a useful tool for routine testing, and selection of virus-free potatoes, especially when a large number of samples are detected.