Development of a Loop-mediated Isothermal Amplification Method for Detection of Environmental Ochrobactrum anthropi

• Main Authors: Chun-Yu Lai, 賴俊宇
• Other Authors: Hsi-Jien Chen
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/42383608821833801099

碩士 === 明志科技大學 === 環境與資源工程研究所 === 102 === Dioxin is one of the most controversial pollutants in 20th century. And, 2,3,7,8-tetrachobenzo-p-dioxin (2,3,7,8-TCDD) has the highest toxicity. The median lethal dose (LD50) testing for rats is 20 μg/kg only. Environmental Protection Administration (EPA) has stipulated the control standards for Polychlorinated dibenzo-p-dioxins/ dibenzofurans (PCDD/DFs). It could not over 1000 ng-TEQ/kg in soil. The common method of quantitating dioxin is using by HRGC/MS. But it could not show the comprehensive influence such as toxicity for bio-hazard. Therefore, the aim of this study is developing a kind of biomarker method to detect or assesses the dioxin in environment. This study has isolated Ochrobactrum anthropi strain BD-1 in dioxin environment and takes its internal transcribed spacer (ITS) as biomarker and designs three primers. Loop-mediated isothermal amplification (LAMP) method is used to amplify ITS, and show the ITS_Spec3 primer has the best specificity. The reaction time for maximal amplification is 60 min, sensitivity is 1 ng. If adding the forward loop primer (FLP) for amplification, the sensitivity is 1 pg which increases 1000 folds. And the reaction time can decrease to 30 min. ITS_Spec3+FLP could detect strain BD-1 in soil and without influences of background DNA. This study successfully builds the linear regression of LAMP reaction time with viable bacterial numbers (colony-forming unit, CFU). It could apply to detect strain BD-1 for pollutant sites in the future.