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KMID : 0613820100200121896
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Journal of Life Science
2010 Volume.20 No. 12 p.1896 ~ p.1901
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Quantitative Real-time PCR using Lactobacilli as Livestock Probiotics
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Choi Yeon-Jae
Kim Seon-Ho
Gu Min-Jeong
Choi Han-Na
Kim Dong-Woon
Cho Sang-Beum
Kim Soo-Ki
Jeon Che-Ok
Bae Gui-Seck
Lee Sang-Suk
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Abstract
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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 10² to 10¹?. 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 10² to 10¹? 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 R² 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 R² were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over 10¹? 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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KEYWORD
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DFM, gene cloning, Lactobacillus, probe, real-time PCR
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