Food Science of Animal Resources (한국축산식품학회지)

Korean Society for Food Science of Animal Resources (한국축산식품학회)
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Rapid and Sensitive Detection of Salmonella spp. by Using a Loop-Mediated Isothermal Amplification Assay in Duck Carcass Sample

오리 도체에서 등온유전자증폭기법을 이용한 Salmonella spp. 신속 고감도 검출 기법 연구

  • Cho, Ae-Ri (College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University) ;
  • Dong, Hee-Jin (College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University) ;
  • Cho, Seongbeom (College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University)
  • 조애리 (서울대학교 수의과대학 공중보건학교실) ;
  • 동희진 (서울대학교 수의과대학 공중보건학교실) ;
  • 조성범 (서울대학교 수의과대학 공중보건학교실)
  • Received : 2013.06.29
  • Accepted : 2013.10.08
  • Published : 2013.10.31
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Abstract

In this study, a rapid and sensitive detection tool for screening Salmonella spp. by using a loop-mediated isothermal amplification (LAMP) assay targeting the genomic sequence of the invA gene was developed. The inclusivity and exclusivity were both at 100% in the analysis, and the limit of detection (LOD) in a pure S. Enteritidis culture suspended in saline was $3.2{\times}10^3$ CFU/mL at 18.17 min ($R^2$ = 0.9446). The LODs of the LAMP assay in buffered peptone water with Salmonella (BPW) and duck carcass swab sample enriched in BPW with Salmonella (BPWS) after 0 and 12 h incubation were $3.2{\times}10^3$ CFU/mL and $3.2{\times}10^0$ CFU/mL, respectively. Comparing the LODs in BPW with those in BPWS, the LAMP assay was less influenced by compounds of duck carcass swab sample than the PCR assay. Sensitivity and specificity of the LAMP assay in 50 duck carcass swab samples enriched in BPW for 6 h were 96% and 84%, respectively, indicating that the LAMP assay is a rapid, simple and sensitive assay, which can be utilized as a potential screening tool of Salmonella spp. in duck carcass sample.

본 연구에서는 Salmonella invA 유전자를 마커로 Salmonella 특이 LAMP primer를 제작하였고, inclusivity 및 exclusivity 실험 결과는 각각 100%로 관찰되었다. 순수 배양된 Salmonella 희석액에서의 검출한계는 18.17분에서 $3.2{\times}10^3$ CFU/mL ($R^2$ = 0.9446)으로 관찰되어 신속 간단하고 민감도가 높아 Salmonella 검출을 위한 방법으로 효과적일 뿐만 아니라 간접적인 정량기법으로서도 활용 가능함을 알 수 있었다. 1차 증균 배지인 BPW에 S. Enteritidis 접종 후 0시간 및 12시간 배양 후 LAMP의 검출한계는 각각 $3.2{\times}10^3$ CFU/mL 및 $3.2{\times}10^0$ CFU/mL으로 관찰되었고, 오리도체 시료가 포함된 BPW에 S. Enteritidis 접종 후 0시간 및 12시간 증균액에서의 LAMP 검출한계는 각각 $3.2{\times}10^3$ CFU/mL 및 $3.2{\times}10^0$ CFU/mL으로 관찰되어 PCR과 비교하였을 때 오리도체시료 성분에 의한 영향이 비교적 낮았음을 알 수 있었다. 또한, 실제 도압장에서 채취한 오리도체시료를 BPW에 6시간 배양한 1차 증균액에 개발한 LAMP기법을 적용한 결과, 96%의 민감도 및 84%의 특이도를 보여 오리도체에서의 Salmonella 신속 스크리닝 기법으로서 효과적으로 활용될 수 있음을 알 수 있었다.

Keywords

References

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