Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Simultaneous Detection of Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus by Multiplex Polymerase Chain Reaction

Multiplex Polymerase Chain Reaction(PCR)법을 이용한 Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus의 다중동시검출

  • 정유석 ((재)대구테크노파크 바이오산업지원센터) ;
  • 정희경 ((재)대구테크노파크 바이오산업지원센터) ;
  • 전원배 (대구경북과학기술원) ;
  • 서화정 (대구경북과학기술원) ;
  • 홍주헌 (대구가톨릭대학교 외식식품산업학부)
  • Published : 2010.04.30
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Abstract

This study was conducted to detect and identify Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella enterica subsp. using simultaneous multiplex polymerase chain reaction (multiplex PCR) assay. 23S rRNA partial gene (S. aureus), tox R gene (V. parahaemolyticus), and inv A gene (S. enterica subsp.) as diagnostic marker gene were suggested, and their amplicon sizes were 482 bp, 368 bp, and 284 bp, respectively. Non specific amplicons by STA-5F/STA-5R primer, ToxR-F/ToxR-R primer, and 139/141 primer were not observed in genomic DNA of pathogen bacteria as Bacillus cereus, Listeria monocytogenes, Escherichia coli, Proteus vulgaris, Streptococcus pyogenes, Candida albicans, and Shigella sonnei. The extracted crude DNA of targeted bacteria was detected as PCR template successfully. The detection limits were $10^5\sim10^4$ CFU/mL and 10 pg of purified genomic DNA of S. aureus, V. parahaemolyticus, and S. enterica subsp. by using simultaneous multiplex PCR.

본 연구는 국내 주요 식중독 원인균인 Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus를 동시에 검출 및 동정할 수 있는 simultaneous multiplex PCR방법을 개발하고자 하였다. S. aureus의 23s rRNA 유전자(482 bp), V. Parahaemolyticus의 toxR 유전자(368 bp), S. enterica subsp.의 invA 유전자(284 bp)를 특이적으로 검출 및 동정할 수 있는 3개 primer set 즉, STA-5F/STA-5R, ToxR-F/ToxR-R, 139/141을 구축하였으며, 그 결과 정제되어진 각 식중독 원인균의 genomic DNA를 template로 하여 세 균주 모두 10 pg까지 다중동시검출이 가능하였다. 생균수(CFU)와 상응되는 검출한계 결과로써 $10^1\sim10^2$ CFU/reaction의 검출한계를 보였으며 이는 즉, S. aureus $6.0\times10^4$ CFU/mL, S. enterica subsp. $9.5\times10^4$ CFU/mL, V. parahaemolyticus $6.1\times10^5$ CFU/mL의 검출한계를 나타내었다. 균체회수부터 agarose gel 상에서 검출 및 동정까지 3~4 hr의 시간 소요로 single tube 반응으로 세 식중독 원인균의 다중동시검출이 가능하였다. 또한 추가적인 연구를 통하여 세 식중독 원인균주의 검출을 위한 향상된 민감도를 가지는 multiplex PCR법 및 real time PCR을 이용한 다중동시검출법 개발을 위한 기초자료로서 활용 가능할 것이라 사료된다.

Keywords

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