Research in Plant Disease 2006;12(2):139-143.
Published online August 30, 2006.
Tomato spotted wilt virus를 위한 간편한 식물바이러스 핵산진단법: Virion Captured/RT-PCR (VC/RT-PCR)
조점덕, 김정수, 김현란, 정봉남, 류기현
Convenient Nucleic Acid Detection for Tomato spotted wilt virus: Virion Captured/RT-PCR (VC/RT-PCR)
Jeom Deog Cho, Jeong Soo Kim, Hyun Ran Kim, Bong Nam Chung, Ki Hyun Ryu
Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RTPCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at 4oC. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at 95oC immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions`` RNAs by heat treatment were used for RT-PCR. Dilution end point of 10-5 from plant``s crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.
Key Words: Extraction buffer, Tomato spotted wilt virus, TSWV, Virion captured (VC)/RT-PCR
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