Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Effective Concentration Method for Applying PCR to Detect Viruses in Water

수계바이러스검출에 PCR을 이용하기 위한 효과적인 농축기법

  • 이승훈 (서울대 자연과학대학 미생물학과) ;
  • 김상종 (서울대 자연과학대학 미생물학과)
  • Published : 1999.03.01
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Abstract

In detecting pathogeuic viruses in water sample, polymerase-chain-reaction (PCR) amplification was uscd. In order lo obtain the intact viral particlc, five concentration techniques were compared and an improved procedure was developed with some modifications. Among them, adsorption-elution~EG precipitation and flocculatio~~iultracentriEugation were more efficient than others with thc detection limit of 10 PFU $ml^{-1}$. By the additional step removing inhibitory compounds for PCR reaction, the purity of the concentrated sample was improved and the detection limit was lowered by one order (to 1 PFU $ml^{-1}$. To examine the availability of the optimized procedure for field surveys, the distributions of enterovirus in Han River were estimated using the novel procedure. Seventy-five percentage (618) of sewagc samples and twenty percentage (2110) of river water samples were positive for enterovirus. These results indicate that adsorption-elutionPEG precipitation by PCR method is useful for the prompt and handy monitoring of viral contaminaiton in water environment and pathogenic viruses are widely distributed in water environments of Seoul.

수계에 분포하는 장바이러스를 검출하기 위해 민감도가 높고 검출시간이 빠른 PCR법을 이용하였다. 바이러스입자를 보다 순수하게 농축하기 위해 몇 가지 바이러스농축법을 비교조사하고 그것의 변형을 통하여 개선된 방법을 확립하였다. 비교한 농축방법중 흡탈착/Polyethyleneglycol(PEG) 침전법과 플록화/촉속원심분리법을 통해 10 PFU $ml^{-1}$ 까지 검출이 가능하여 다른 방법에 비해 효과적임이 판명되어Te. Sephadex resin을 이용한 부가적인 시료정제 과정을 통해 시료의 순도를 높일 수 있어검출한계를 10배정도 낮추었다. 서정된 검출법을 통해 한강수계에서 장바이러스의 분포를 조사한 결과 조사한 시료중 한강의 지천에서는 75%, 본류에서는 20%의 시료에서 바이러스가 검출되었다. 본 조사결과 흡탈착/PEG 침전법-PCR 검출기법이 수계의 바이러스 오염도를 간편하고 신속하게 검출하는데 매우 유용하였으며 국내 수계에도 바이러스 오염이 광범위함을 확인하였다.

Keywords

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