Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Quantitative Real-time PCR using Lactobacilli as Livestock Probiotics

Real-time PCR을 이용한 가축생균제용 유산균 정량분석

  • Choi, Yeon-Jae (Department of Animal Science & Technology, Sunchon National University) ;
  • Kim, Sun-Ho (Department of Animal Science & Technology, Sunchon National University) ;
  • Gu, Min-Jeong (Department of Animal Science & Technology, Sunchon National University) ;
  • Choe, Han-Na (Depart of Biology, Sunchon National University) ;
  • Kim, Dong-Un (National Institute of Animal Science, RDA) ;
  • Cho, Sang-Bum (Division of Animal Life Science, Konkuk University) ;
  • Kim, Su-Ki (Division of Animal Life Science, Konkuk University) ;
  • Jeon, Che-Ok (Department of Life Sciences, ChungAng University) ;
  • Bae, Gui-Seok (Department of Animal Science & Technology, ChungAng University) ;
  • Lee, Sang-Seok (Department of Animal Science & Technology, Sunchon National University)
  • 최연재 (순천대학교 동물자원과학과) ;
  • 김선호 (순천대학교 동물자원과학과) ;
  • 구민정 (순천대학교 동물자원과학과) ;
  • 최한나 (순천대학교 생물학과) ;
  • 김동운 (농촌진흥청 국립축산과학원) ;
  • 조상범 (건국대학교 동물생명과학부) ;
  • 김수기 (건국대학교 동물생명과학부) ;
  • 전체옥 (중앙대학교 생명과학과) ;
  • 배귀석 (중앙대학교 동물자원과학과) ;
  • 이상석 (순천대학교 동물자원과학과)
  • Received : 2010.11.20
  • Accepted : 2010.11.23
  • Published : 2010.12.30

Abstract

This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$. Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.

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Acknowledgement

Supported by : 농촌진흥청

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