Immunomagetic separation과 RT-PCR 방법을 이용한 굴에서의 Feline calicivirus 신속검출
Rapid detection of Feline calicivirus in oyster using Immunomagetic separation and RT-PCR method
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초록
우리나라에서도 노로바이러스에 의해 발생하는 식중독이 급격히 증가하는 추세를 나타내고 있다. 그러나 식품에서의 노로바이러스의 검출 기술은 미흡하여 식중독 발생시 역학조사가 어려운 실정이다. 따라서 본 연구에서는 IMS와 RT-PCR을 사용하여 굴에 오염된 노로바이러스의 신속검출법을 개발하였다. 노로바이러스의 대체모델로서 같은 Caliciviridae 과에 속하며 배양이 가능한 feline calicivirus(FCV)를 이용하고 IMS 법과 CPT(chloroform, polyethylene glycol, Tristween)법을 사용하여 굴에서의 FCV 검출효율을 비교하였다. 굴 10g에 FCV를 접종하고 배양시킨 다음 PBS 90 mL를 첨가하여 균질화한 후, chloroform 처리하여 IMS를 실시하였으며, CPT에서는 chloroform 처리 후 polyethylene glycol과 Tristween을 처리한 후 RT-PCR을 통하여 검출여부를 확인하였다. IMS의 검출한계는 5.7×103 PFU/mL로 comtrol 농도인 5.7×101 PFU/mL보다 낮았다. 굴에서 CPT와 IMS에 의한 RT-PCR의 검출한계는 각각 1.05×104 PFU/g과 3.77×103 PFU/g였다. 식품성분중의 RT-PCR inhibitor의 존재하에서 IMS의 경우가 CPT보다 더 높은 검출효율을 나타내었다. 본 연구에서 개발된 신속검출법은 굴에서 FCV와 NV를 검출하는데 효율적으로 적용될 수 있을 것으로 생각된다.검색어 - 노로바이러스, Feline calicivirus, Immunomagnetic 분리, RT-PCR, 굴
Abstract
Foodborne illness caused by Noroviruses(NV) are increasing rapidly in Korea. In this study, we developed a rapid and simple detection protocol for NVs contaminated in oyster using immunomagnetic separation(IMS) and RT-PCR. As a surrogate model for NVs, the culturable feline calicivirus (FCV) that belongs to the same Caliciviridae family was used. Detection efficiency for FCV in oyster was compared with IMS and CPT(chloroform, polyethylene glycol and Tris·tween) Direct homogenization of 10 g sample of whole oyster in 90 mL PBS provided the simplicity needed in the actual field of food product examination. First, IMS was immediately performed after chloroform treatment. In CPT, polyethylene glycol and Tris·tween treatment were performed after chloroform treatment, and FCV was detected through RT-PCR. IMS detection limit (5.7×103 PFU/mL) was lower than control detection as 5.7×101 PFU/mL. The detection limits of RT-PCR for FCV by CPT and IMS in oyster were 1.05×104 PFU/g and 3.77×103 PFU/g, respectively. In the presence of RT-PCR inhibitors from food materials, detection efficiency of IMS was higher than CPT. The rapid and simple detection protocol developed in this study could be efficiently applied to detect FCV and probably NV from in oyster.
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