One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacterium and Virus Occurring on Brassicaceae Crop Seeds

Jeong, Kyu Sik; Soh, Eun Hee

Research in Plant Disease 2011;17(1):52-58.
Published online April 30, 2011.

십자화과 작물 종자에서 종자전염 세균 및 바이러스 동시 검출을 위한 One-step Multiplex RT-PCR 방법

정규식, 소은희

One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacterium and Virus Occurring on Brassicaceae Crop Seeds

Kyu Sik Jeong, Eun Hee Soh

Abstract

The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc) and Lettuce Mosaic Virus (LMV). Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage), two pairs of specific primer (LMV-F/R, Xcc-F/R) were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/). The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties), lettuce (50 varieties), radish (20 varieties), chinese cabbage (20 varieties) and cabbage (20 varieties), LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.

Key Words: Brassicaceae crop, Mutliplex RT-PCR, Seed pathogens