Research in Plant Disease 2012;18(2):73-79.
Published online June 30, 2012.
보문 : 밤나무 잉크병균, Phytophthora katsurae의 검출을 위한 Duplex PCR
이동현 ( Dong Hyeon Lee ) , 이선근 ( Sun Keun Lee ) , 김혜정 ( Hye Jeong Kim ) , 이상현 ( Sang Hyun Lee ) , 이상용 ( Sang Yong Lee ) , 이종규 ( Jong Kyu Lee )
 
A Duplex PCR for Detection of Phytophthora katsurae Causing Chestnut Ink Disease
Dong Hyeon Lee, Sun Keun Lee, Hye Jeong Kim, Sang Hyun Lee1, Sang Yong Lee and Jong Kyu Lee*
Department of Forest Environment Protection, Kangwon National University, Chuncheon 200-701, Korea
1Division of Forest Insects and Diseases, Korea Forest Research Institute, Seoul 130-712, Korea
Received: May 31, 2012   Revised: June 08, 2012   Accepted: June 08, 2012
Abstract
Phytophthora katsurae is a fungal pathogen responsible for chestnut ink disease. We designed two duplex primer sets (SOPC 1F/1R+KatI 3F/5R, SOPC 1-1F/1-1R+KatI 3F/5R) to detect P. katsurae. SOPC 1F/1R and SOPC 1-1F/1-1R primer pairs were designed for sequence characteristic amplification regions (SCAR) marker, and KatI 3F/5R primer pair was used for P. katsurae-specific primer designed from internal transcribed spacer (ITS) region. To assess the sensitivity of duplex PCR, genomic DNA was serially diluted 10-fold to make the final concentrations from 1 mg/ml to 1 ng/ml. The sensitivity for two primer sets were 1 μg/ml and 100 ng/ml, respectively. To find detection limits for zoospores of P. katsurae, each zoospore suspension was serially diluted 10-fold to make the final concentrations from 1× 106 to 1 × 102 cells/ml, and then DNA was extracted. The limits of detection for all of two primer sets were 1 × 105 cells/ml. All of two primer sets were specific to P. katsurae in PCR detection and did not produce any P. katsurae-specific PCR amplicons from other 16 Phytophthora species used as the control. This study shows that duplex PCR using two primer sets might be a useful tool for rapid and efficient detection of P. katsurae.
Key Words: Chestnut ink disease, Duplex PCR, Phytophthora katsurae


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