| Summary: | 碩士 === 大葉大學 === 分子生物科技學系碩士班 === 102 === In this study, we use multiplex Reverse-transcription polymerase chain reaction (RT-PCR) technique simultaneously detected two viroid species in grapevines. Twenty five leaf samples with yellowing or mosaic symptoms were collected from difference vineyards at Changhua county, Taiwan during May to June, 2013. Specific primer pairs for the detection of Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2), and Citrus exocortis viroid (CEVd) were selected from previous reports or were newly designed according to the sequences obtained from NCBI GenBank. Single one-step RT-PCR analyses were performed and found that all the collected samples were infected by HSVd and 16% of them were infected by GYSVd-1. The amplicons from RT-PCR were cloned into TA vector and the sequences analysis revealed that HSVd-DY contains 301 nt, while GYSVd-1-DY contains 367 nt. Comparison of the sequence identity of HSVd-DY with other HSVd from GenBank ranged from 99.0% to 82.9%. While the sequence identity between GYSVd-1-DY with other GYSVd-1 ranged from 99.7% to 85.9%. A phylogenetic tree derived from the complete sequence of GYVd-1 indicated that GYVd-1-DY was closely related to a China isolate (JF746188). Multiplex RT-PCR amplification was successfully performed to identify HSVd and GYSVd-1 from grapevine samples. The established of multiplex RT-PCR analyses possess a highly sensitivity, rapid, and low-cost strategy for detection of difference viroids in vineyards.
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