The Establishment Of Real-Time RT-PCR And RT-LAMP Techniques For The Detection Of Three Viruses Infecting Pear Plants

Apple stem grooving virus(ASGV), Apple stem pitting virus(ASPV) and Apple cholortotic leaf spot virus(ACLSV) are most common virus infecting pear. Pear infected by single virus will not show obvious symptoms. However, if hosts were infected by two or three virus, some pear leaves will show faded green spot, vein yellow, leaf deformity and curl and other obvious symptoms. Currently, the cultivation of virus-free pears is the most effective way to prevent and control plant virus disease. Therefore, the detection and diagnosis of virus play a significant importance in virus disease. Moreover, the establishment of high sensitivity, fast, high-throughput detection provided technical support for the cultivation of virus-free seedlings. This study established a detection system with reverse transcriptional real time fluorescent quantitative and reverse transcription loop-mediated isothermal amplification for the three viruses in pears. The results are summarized as follows:1. In this research, specificity qRT-PCR primers are designed according to cp genes sequence of ASGV, ASPV and ACLSV. Finally, ASGV-F6/R6, ASPV-F8/R8 and ACLSV-f5/r5 were filtered as stable primer in the test.By optimizing the conditions, the primer concentration and annealing temperature were confirmed, which were 0.15 pmol/μl and 58℃, respectively. The study found that primers content and annealing temperature have major impact on qRT- PCR. In vitro pear plants, the lowest concentration of cDNA of the ASGV, ASPV and ACLSV were 4×10-3ng/μl, the sensitivity of this method is 103,103 and 102 times than RT-PCR.2. qRT-PCR system was applied to detect virus infecting vitro plant that the result showed that positive rate of ASGV, ASPV and ACLSV is 68.2%,50.0% and 4.5%, compared with RT-PCR, the detection rate of ASGV and ASPV is 9.1% higher, but the ACLSV is quite. And the field samples of pear is 35.0%,7.5% and 5.0%, the detection rate of ASGV and ASPV is 5.0% higher than RT-PCR, the ACLSV is consistent. LAMP primers were designed 5 and 6 groups for detection of ASPV and ACLSV, we select of the positive pear leaves as materials, which two sets were selected that were ACLSV-6-F3/R3, ACLSV-6-FIP/BIP and ASPV-5-F3/B3, ASPV-5-FIB/BIP at last. Finally, LAMP reaction system is established by optimizing the concentration of dNTP, Mg2+ betaine, primer ratio, reaction time and reaction temperature. Using of RT-LAMP detection of ACLSV and ASPV, the minimum concentration of total of RNA is 10-4 ng/μl and 103 ng/μl, the sensitivity of this method were 104 times than RT-PCR. PCR products were used to the dye of S YBR Green I direct observation and agarose gel electrophoresis, the results showed that the dye is added prior to the reaction product can be directly observed, the sensitivity is 10 times higher than agarose gel electrophoresis.4. When detect the randomly selected of 20 pear plantlets, the detection rate of ASPV and ACLSV is 45.0% and 15.0%, and twenty-two field samples of pears is 40.9% and 36.4%, respectively. The detection rate of the pear plantlets and the field samples of ASPV is 5.0% and 4.5% higher than RT-PCR, however, the ACLSV is consistent and 4.6% higher, respectively.