Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Establishment of Sample Preparation Method for PCR Detection of Clostridium perfringens from Agricultural Products

PCR 법을 이용한 농산물 중 Clostridium perfringens 검출을 위한 전처리법 확립

  • Choi, Song-Yi (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Seo, Min-Kyoung (Food Safety Risk Assessment Division, Natioanal Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Yoon, Jae-Hyun (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Rajalingam, Nagendran (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Hwang, Injun (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Kim, Se-Ri (Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development Administration)
  • 최송이 (농촌진흥청 국립농업과학원 유해생물팀) ;
  • 서민경 (식품의약품안전처 식품식품위해평가과) ;
  • 윤재현 (농촌진흥청 국립농업과학원 유해생물팀) ;
  • 나겐드란 라잘링감 (농촌진흥청 국립농업과학원 유해생물팀) ;
  • 황인준 (농촌진흥청 국립농업과학원 유해생물팀) ;
  • 김세리 (농촌진흥청 국립농업과학원 유해생물팀)
  • Received : 2020.10.10
  • Accepted : 2020.11.26
  • Published : 2021.02.28
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Abstract

This study was undertaken to compare the efficacy of different sample preparation (stomaching, pulsifying, and sonication) and DNA extraction methods (boiling and commercial kit) for detection of enterotoxin-producing Clostridium perfringens from produce by polymerase chain reaction (PCR). Each produce type was inoculated at concentrations of 102, 103, 104, 105, 106, and 107 spores/g. Produce inoculated with spores was treated with three sample preparation methods, and DNA was extracted by boiling method and a commercial kit, followed by PCR. The detection limit of stomached samples was lower than that of pummeled and sonicated samples by 10-100 times. Moreover, the DNA extraction efficiency of the commercial kit was found to be superior to that of boiling. In particular, the PCR efficiency of cherry tomato and perilla leaf samples was greatly affected by sample preparation and DNA extraction method. These data suggest that DNA extraction with a commercial kit after pulsification is an optimum sample preparation method for detection of C. perfringens by PCR.

본 연구에서는 PCR법을 이용하여 농산물 중 enterotoxin 생성 Clostridium perfringens를 신속 분석할 수 있도록 전 처리법을 확립하고자 수행하였다. 이를 위하여 C. perfringens 포자를 상추, 토마토, 고추, 들깻잎에 102, 103, 104, 105, 106, 107 spore/g 농도로 포자를 접종하였다. 포자가 접종된 농산물들은 pulsifier, stomacher, sonicator로 처리하고 boiling법 혹은 상용화 된 kit로 DNA를 추출한 후 PCR법을 수행하고 검출한계를 비교하였다. 그 결과, 3가지 전처리법에 있어서는 pulsifier가, DNA 추출에 있어서는 상용화된 DNA 추출 kit를 활용하는 것이 농산물 중 C. perfringens의 검출한계를 10-100배 낮출 수 있었다. 특히 들깻잎, 방울토마토처럼 전처리 방법에 따른 탁도의 변화가 큰 농산물은 전처리법과 DNA 추출법이 PCR 반응에 미치는 영향이 큰 것으로 나타났다. 따라서 본 연구 결과를 통해 볼 때 PCR법을 이용한 농산물 중 C. perfringens를 검출하는데 있어 검출감도를 높이기 위해서는 pulsifier를 이용하여 전처리하고 상용화된 DNA 추출 kit를 사용하는 것이 적절한 것으로 판단된다.

Keywords

References

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