Journal of the Korean Chemical Society (대한화학회지)

Korean Chemical Society (대한화학회)
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Diagnosis of Bovine Theileriosis by Direct PCR and Electrophoresis from Whole Blood Without DNA Extraction

DNA 추출없이 전혈을 이용한 PCR-전기영동법에 의한소의 타일레리아병 진단

  • Seong Ho Kang (Department of Chemistry, Chonbuk National University) ;
  • Sangmin Jang (Department of Chemistry, Chonbuk National University) ;
  • Joon-Seok Chae (Bio-safety Research Institute, Chonbuk National University) ;
  • Yongseong Kim (Division of Chemistry and Chemical Engineering, Kyungnam University)
  • Published : 2003.04.20
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Abstract

We have developed a direct polymerase chain reaction (PCR) and electrophoresis method for the diagnosis of bovine theileriosis from whole blood DNA analysis without DNA extraction. The technique empolyed a FoLT (formamide, low temp.) technique and was utilized in the diagnosis of bovine theileriosis. Formamide solubilize the blood cells, and the lowered incubation temperatures reduced protein coagulation. 100-200 nL of whole blood and PCR reagents were introduced directly into a PCR tube. After the amplification, the PCR product (816-bp DNA) was introduced into the electrophoresis system. The results of this analysis were consist with those obtained using purified DNA.

소의 타일레리아병 진단을 위해 DNA 추출과정 없이 전혈에서 바로 중합효소연쇄반응(PCR)을 통하여 T. buffeli(buffeli/orientalis/sergenti)의 16S rRNA 유전자 단편을 증폭시킨 뒤, 증폭된 DNA를 전기영동법으로 분석하는 방법을 개발하였다. 특이유전자 단편의 증폭을 위해 formamide를 사용하여 혈액세포를 용해시켰으며, 단백질의 응고를 줄이기 위해 낮은 반응온도를 사용하는 FoLT(Formamide Low Temp.) PCR법을 이용하였다. 전혈 100-200 nL를 바로 PCR 증폭에 사용하였으며, PCR 산물(816-bp DNA)은 전기영동법으로 분석하였다. 본 결과는 T. buffeli에 감염된 소의 혈액으로부터 정제된 DNA를 사용하여 얻은 실험결과와 잘 일치하였다.

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