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Development of Ultra-Rapid Multiplex PCR Detection against 6 Major Pathogens in Honeybee

꿀벌 6종 주요 병원체에 대한 초고속 다중 PCR 검출법의 개발

  • Lim, Su-Jin (Department of Life Science, College of Natural Science, Kyonggi University) ;
  • Kim, Jung-Min (Department of Life Science, College of Natural Science, Kyonggi University) ;
  • Lee, Chil-Woo (Korea Honey Bee Disease Institute) ;
  • Yoon, Byoung-Su (Department of Life Science, College of Natural Science, Kyonggi University)
  • 임수진 (경기대학교 자연과학대학 생명과학과) ;
  • 김정민 (경기대학교 자연과학대학 생명과학과) ;
  • 이칠우 (한국꿀벌질병연구소) ;
  • 윤병수 (경기대학교 자연과학대학 생명과학과)
  • Received : 2016.11.30
  • Accepted : 2017.04.05
  • Published : 2017.04.30

Abstract

PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than $10^3$ molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.

Acknowledgement

Supported by : 농림수산식품기술기획평가원