马铃薯六种主要病毒通用RT-PCR检测体系的建立

摘要

马铃薯X病毒（PVX）、马铃薯Y病毒（PVY）、马铃薯S病毒（PVS）、马铃薯卷叶病毒（PLRV）、马铃薯M病毒（PVM）和马铃薯A病毒（PVA）是马铃薯生产田中发生率较高、危害较严重的病毒，因此，建立特异性强、灵敏度高、检测速度快的RT-PCR分子检测体系对保障马铃薯种薯质量具有非常重要的意义。试验筛选了6种病毒的特异引物、优化了PCR部分的试剂以及确定了退火温度。结果表明，当Mg2+浓度为2.6 mmol/L、dNTPs浓度为0.1 mmol/L、Taq DNA聚合酶浓度为0.03 U/μL，以及退火温度为55.5℃时反应体系最稳定。最终，建立了一套通用RT-PCR检测体系，只需要变换每种病毒的引物，就可以实现对PVX、PVY、PVS、PLRV、PVM和PVA病毒的RT-PCR检测。每种病毒检测灵敏度均能达到pg以上。%The higher incidence and more serious viruses in potato fields are potato virus X (PVX), potato virus Y (PVY), potato virus S (PVS), potato leafroll virus (PLRV), potato virus M (PVM), and potato virus A (PVA), so it is very important to guarantee the quality of seed potato by establishment of RT-PCR molecular detection system with specificity, high sensitivity and fast detection speed for these viruses. In this research, the primer for each virus was screened, PCR reagents were optimized, and annealing temperature was determined. The results indicated that when the concentration of Mg2 + was 2.6 mmol/L, dNTPs was 0.1 mmol/L, Taq DNA polymerase was 0.03 U/μL, and annealing temperature was 55.5℃, the reaction system was the most stable. Finally, a universal RT-PCR detection system was established, which only need to change primer for each virus when the RT-PCR detection for PVX, PVY, PVS, PLRV, PVM and PVA was needed. The detection sensitivity of each virus could reach the level of pg.