Research On Detection Of Plant Viroid Using Magnetic Particles Combined With RT-PCR And Real-time Quantitative RT-PCR

The viroid has the specificities with stronger infection capability and longer latency, can infects many kinds of plants. Because of viroid was difficult to be detected and controled, viroid has serious economic implications for industrial crops. In recent years, molecular biology techniques were widely used in the detection of plant viriod, because of their rapid, efficient and sensitivity. The specificity and sensitivity of molecular detection methods are largely determined by the quantity and quality of nucleic acids extract from plant tissue, Since nucleic acids are the foundation of a cDNA library, hybridization, gene cloning, RT-PCR and other operations.This research established a sensitive and high stability detection method of plant viroid based on the extraction of nucleic acid using Nanometer magnetic particles, combined with ordinary RT-PCR/RT-qPCR detection method.Firstly, the total nucleic acid was ectracted by employing magnetic particles. It is a very promising method. The method had the advantages of simplicity, high stability and good reproducibility for the MNP’s large specific surface area, easily modified and Operability. A good system was established for detection of plant viroids by combining with RT-PCR/RT-qPCR.Secondly, the experiment conditions about the size and time of centrifugal force, the amount of MNP and ethanol, the time and temperature of combination and acids release, cleaning times were optimized. Since the detection system combining magnetic particles with RT-PCR/RT-qPCR was completed. When the supernatant is200μl, the optimal conditions are2mg magnetic particles,100μl ethanol,2-5min bonding time, washing2-4times and washing off2-5min.Lastly, the detection method was applied for detection of Hop latent viroid(HLVd) and Apple fruit crinkle viroid (AFCVd). The results showed that the detection system combining magnetic particles with RT-PCR/RT-qPCR is sensitive, specificity and accurate.In conclusion, the MNP combining with RT-PCR/RT-qPCR techniques can detected plant viroids effectively. The method meets the practical needs of inspection and quarantine. It can be used in entry-exit inspection and quarantine phytosanitary and diagnosis of plant viroids in research institutions. It’s important for improving the level of inspection and quarantine, and the export clearance rate of the plant and its related products.