A Multiplex RT-PCR Assay for Detecting Three Pathogens Infecting Citrus Plants
[Objective]A multiplex RT-PCR assay to simultaneously detect citrus yellow vein clearing virus(CYVCV),citrus tristeza virus(CTV),and hop stunt viroid(HSVd)was developed.[Method]Multiplex RT-PCR primers were designed,and their specificity was analyzed.The optimal concentration ratio,annealing temperature and sensitivity of primers were determined.Moreover,citrus samples from Fujian region were detected by the established multiplex RT-PCR.[Result]The optimized concentration ratio of CYVCV-F/R,CTV-F/R,and HSVd-F/R primers was 1:1:2,and the annealing temperature at 52.9℃for the assay.The assay displayed a sensitivity at 10-2 dilution and the positive detections on CYVCV of 47.1%,on CTV of 56.7%,and on HSVd of 22.9%on 157 detected samples.[Conclusion]A rapid,accurate,applicable multiplex RT-PCR method for CYVCV,CTV,and HSVd detections was successfully established.
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