Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Methods of in situ PCR to Retain the Amplification Products Inside the Cells

원위치 중합효소 연쇄반응에서 증폭산물의 세포내 보존을 위한 방법들

  • 이재영 (목포대학교 자연과학대학 생물학과)
  • Published : 2001.12.01
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Abstract

Highly effective polymerase chain reaction (PCR) often brings about false positivity caused by contamination of the sample with target nucleic acids. To solve this problem, in situ PCR (ISPCR) has been developed and applied onto various tissue sections and suspension cultures. With combination of PCR and in situ hybridization, this method amplifies the nucleic acid targets in situ and detect the amplified products inside the cells over the background of various cell types. In order to amplify the nucleic acid targets inside the cells, permeabilisation of a sample is required for the entry of amplification reactants into a cell. Treatments of a sample for the purpose allow not only the entry of reactants into the cell but also the exit of amplification products out of the cell. As a means to reduce the leakage of the amplification products, two methods were applied to suspension cultures of HIV-infected Molt/LAV and U 1.1 cells, in which modified, tailed primers produced long linear amplificants whereas biotinylated dUTP instead of dTTP did bulky products.

감염성 바이러스의 발생은 세계적인 현상으로 어린이는 물론 성인의 건강을 위협하고 있는 실정이다. 1998년 -2000년 사이에 부산지역 바이러스성 전염병 유행예측사업의 과정에서 소화기계 바이러스가 탐색되었다. 의심되는 환자의 대변 및 뇌척수액, 인후가검물에서 세포배양, Latex 응집반응, 간접면역형광항체법, 전자현미경 관찰 등을 행하여 바이러스를 확인하였다. 총 검체 중에서 바이러스의 확인 율은 12.5% 이었다. 이 과정을 통하여 3 사례의 장 adenovirus 및, 23 사례의 echovirus, 31 사례의 coxsackievirus, 36 사례의 rotavirus, 45 사례의 small round structured virus (SRSV), 7 사례의 poliovirus가 확인되었다. 확인된 주요 혈청형으로는 장 adenovirus 41형 및 echovirus 6, 9, 11, 25, 30형, coxsackievirus B2, B3, B4, B6 형 등이 탐색되었다. 각 바이러스의 월별 발생별로는 SRSV는 12월에서 다음해 4월 사이, echovirus와 coxsackievirus는 4월에서 10월 사이에, rotavirus는 1월에서 4월 사이에 각각 분리 율이 높았다. 전자현미경 관찰에서는 30-80 nm의 작은 크기의 바이러스들이 확인되었다.

Keywords

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