[논문]RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

Lee, Jong-Seung; Cho, Won-Kyong; Choi, Hong-Soo; Kim, Kook-Hyung

doi:10.5423/ppj.2011.27.3.291

RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses 원문보기

The plant pathology journal, v.27 no.3, 2011년, pp.291 - 296

Lee, Jong-Seung (Department of Agricultural Biotechnology and Plant Breeding and Genomics Institute, College of Agriculture and Life Sciences, Seoul National University) , Cho, Won-Kyong (Department of Agricultural Biotechnology and Plant Breeding and Genomics Institute, College of Agriculture and Life Sciences, Seoul National University) , Choi, Hong-Soo (Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration) , Kim, Kook-Hyung (Department of Agricultural Biotechnology and Plant Breeding and Genomics Institute, College of Agriculture and Life Sciences, Seoul National University)

Abstract ▼ AI-Helper

In order to detect quarantine plant viruses, we developed reverse transcription-polymerase chain reaction (RT-PCR) primer pairs for five single-stranded (ss) plant RNA viruses that are not currently reported in Korea but could be potential harmful plant viral pathogens. Three viruses such as Chilli veinal mottle virus (ChiVMV), Colombian datura virus (CDV), and Tobacco etch virus (TEV) belong to the genus Potyvirus while Chrysanthemum stem necrosis virus (CSNV) and Iris yellow spot virus (IYSV) are members of the genus Tospovirus. To design RT-PCR primers, we used reported gene sequences corresponding to the capsid protein and polyprotein for ChiVMV, CDV, and TEV while using nucleocapsid protein regions for CSNV and IYSV. At least two different primer pairs were designed for each virus. Fifteen out of 16 primer pairs were successfully applied in detection of individual quarantine virus with high specificity and efficiency. Taken together, this study provides a rapid and useful protocol for detection of five quarantine viruses.

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문제 정의

In a series of detecting quarantine plant viruses, five additional single-strand RNA viruses causing significant economic problems to many vegetables and flowering plants were further chosen in this study. These viruses include Chilli veinal mottle virus (ChiVMV; Hwang et al.

제안 방법

The imported samples were either dried plant materials or frozen total nucleic acids deposited at −20℃. All processes related to the import of quarantine viruses were done after permission from NPQS and all experiments were carried out in restricted area to prevent virus outflow. Total RNAs derived from virus infected plant tissues as well as healthy leaves of Nicotiana benthamiana and tomato were prepared by extraction with Trizol Reagent^® (Molecular Research Center, Inc.
, 2011; Park and Kim, 2004). In this study, a set of new primers for the detection of five nonreported plant viruses were designed and employed for RTPCR detection. The specificity of each newly designed primer pairs was also evaluated by RT-PCR assay.
To optimize annealing temperature for individual primer pair, gradient PCR was conducted at three different temper atures including 52℃, 55℃, and 57℃ (Fig. 2). Interestingly, as annealing temperature goes up, the amount of amplified PCR was increased.

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