Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Enhancing Conventional PCR for Detection of Erwinia amylovora

화상병원세균 검출을 위한 Conventional PCR 향상

  • Hyun Ju Choi (Interdisciplinary Program in Smart Agriculture, Kangwon National University) ;
  • Yeon Ju Kim (Interdisciplinary Program in Smart Agriculture, Kangwon National University) ;
  • Jeong Ho Choi (Interdisciplinary Program in Smart Agriculture, Kangwon National University) ;
  • Dong Hyuk Choi (Interdisciplinary Program in Smart Agriculture, Kangwon National University) ;
  • Duck Hwan Park (Interdisciplinary Program in Smart Agriculture, Kangwon National University)
  • 최현주 (강원대학교 스마트농업전공) ;
  • 김연주 (강원대학교 스마트농업전공) ;
  • 최정호 (강원대학교 스마트농업전공) ;
  • 최동혁 (강원대학교 스마트농업전공) ;
  • 박덕환 (강원대학교 스마트농업전공)
  • Received : 2024.08.23
  • Accepted : 2024.09.19
  • Published : 2024.09.30
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Abstract

Polymerase chain reaction (PCR) methods, including conventional PCR (cPCR) and quantitative real-time PCR (qRT-PCR), with both plasmid- and chromosome-targeting primers, are currently the most reliable methods for detecting Erwinia amylovora due to their high sensitivity and specificity. Despite qRT-PCR's quantitative advantage, cPCR remains an attractive method to detect this bacterium in initial screenings of suspected host plants, as it is cost-effective and does not require skilled personnel in well-equipped laboratories. This study aimed to significantly improve cPCR robustness via application of bovine serum albumin (BSA) as a PCR facilitator, with a modified EaF/R primer pair, as previously reported. Experiments have shown that simple supplementation with BSA (10 mg/ml) enhances cPCR reactions using templates such as genomic DNA, bacterial cells, and infected symptomless host organs, including immature apple fruits and seedlings, with EaF/R primers. The cPCR method described in this study is simple, specific, and reliable, and can be applied in routine assays to diagnose fire blight.

현재까지 화상병원세균 진단에서 가장 유용하게 사용되는 방법은 이들 병원세균의 플라스미드 또는 염색체 기반 특이 프라이머를 이용한 일반 PCR 및 정량 PCR 방법이 가장 민감하고 특이성 높은 방법으로 여겨지고 있다. 이들 PCR 중, 일반 PCR 방법은 가격적 측면과 연구자의 기능적 숙련도 및 고가의 장비를 요구하지 않는 다는 측면에서 활용성이 유지되고 있다. 따라서 본 연구에서는 화상병원세균 검출에서의 일반 PCR 반응의 증진효과를 나타내기 위한 일반 PCR 프라이머 및 촉진제를 선발하고자 하였다. 이에 새롭게 제작된 EaF/R 프라이머와 PCR 반응액에 BSA을 10 mg/ml 농도로 추가 첨가한 경우가 화상병원세균 DNA 및 세균 자체 그리고 감염 후 무증상 시료 추출액을 주형으로 하는 일반 PCR 반응 모두를 증진시키는 결과를 확인하였다. 따라서 새롭게 제작된 EaF/R 프라이머 및 BSA 추가 첨가 방법은 최적 발생시기에 집중되는 대량 시료로부터 초기 화상병 이병시료를 선발할 수 있는 일반 PCR 방법의 효율성을 증대시켜 정밀 검진 대상시료의 최소화를 통한 확진시간의 단축 등에 활용될 수 있을 것으로 사료된다.

Keywords

Acknowledgement

This research was supported by Rural Development Administration (PJ014219032021), Republic of Korea.

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