Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Detection and quantitation of Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli O157:H7 by droplet digital PCR

Droplet Digital PCR을 이용한 Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium과 Escherichia coli O157:H7의 검출 및 정량

  • Received : 2016.05.25
  • Accepted : 2016.07.07
  • Published : 2016.10.31
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Abstract

In this study, we investigated the possibility of Droplet digital PCR (ddPCR) for detection of foodborne pathogens. ddPCR combines partitioning of PCR reactions into several thousands or millions of individual droplets in a water-oil emulsion, and counting of positive PCR reaction using flow cytometry. Four species of foodborne pathogens, Bacillus cereus, Staphylococcus aureus, Salmonella Typhimurium and Escherichia coli O157:H7, were used to quantify the target sequence with each of the designed primers and double stranded DNA-binding Evagreen dye. All tested foodborne pathogens showed a detection limit ranging from $100fg/{\mu}L$ to $10ng/{\mu}L$. It was concluded that ddPCR could be used to detect very low concentrations of foodborne pathogens from complex food matrices. For multi-detection of target pathogens, we also tested the samples using multiplex ddPCR and obtained successful results.

본 논문에서는 E. coli O157:H7, S. Typhimurium, S. aureus와 B. cereus에 대한 ddPCR의 검출 효율과 검출한계를 측정하였으며 동시 검출을 위한 multiplex 검출 가능성을 타진하였다. ddPCR은 PCR mix를 포함한 시료를 15,000-20,000개의 droplet으로 분할하여 PCR 하는 방법으로 droplet reader를 이용하여 droplet의 형광 신호를 계수하였다. 식중독 세균의 표적 DNA를 검출하기 위해 2가지 색의 형광 probe (TaqMan)를 제작하였다. ddPCR은 표적 유전자의 형광 신호를 $100fg/{\mu}L$부터 $10ng/{\mu}L$까지의 DNA를 검출 할 수 있었다. 이후에 두 종류의 식중독 세균에서 프라이머 농도를 달리하여 표적 DNA 증폭 크기의 분포가 서로 다르게 구별할 수 있음을 확인하여 multiplex PCR의 가능성이 있음을 알 수 있었다. ddPCR은 비교적 낮은 검출한계를 가지기 때문에 식품에 적은 농도로 존재하는 식중독 세균의 검출에 활용이 가능할 것으로 생각되었다. 또한 식품의 전처리 조건 확립과 반응조건 확립을 통하여 향후 복잡한 matrix effect를 가지는 식품에서 극 미량의 균 검출도 가능할 것으로 생각되었다.

Keywords

References

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