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Studies on a Real-time Turbidimeter-based Loop-mediated Isothermal Amplification for Detection of Roundup Ready Soybean

Author: YuanZuoNa
Tutor: WangZongDe；ShiLei
School: Jiangxi Agricultural University,
Course: Botany
Keywords: Roundup Ready Soybean Loop-mediated isothermal amplification Real - time turbidity method Rapid detection
CLC: S565.1
Type: Master's thesis
Year: 2011
Downloads: 27
Quote: 0
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Abstract

Transgenic technology to get an unprecedented development, a large number of crops by transgenic technology to obtain a variety of new traits, transgenic plants as the raw material of the manufacturing and processing of various transgenic food is to expand its share of the food market share. Although this change so that the world food shortage, lack of resources have been alleviated to some extent, but the safety of genetically modified plants should not be overlooked. Transgenic plants security in the short period of time can not be determined, therefore, the world of genetically modified crops and their derivatives with a labeling system for management. Soybean as a major importer of crops for which GMO detection is particularly important in the transgenic soybeans, glyphosate-resistant soybean accounted for 95%. Established a new rapid test loop-mediated isothermal expansion growth method (loop-mediated isothermal amplification, the LAMP) real-time turbidity assay detection of glyphosate-resistant soybeans, the main content is as follows: 1, mainly for glyphosate-resistant transgenic soybean 5 - shikimate 3 - phosphate synthase (cp4-epsps) gene of a conserved sequence of six regional designed two pairs of primers for detecting the suitability and stability of the LAMP reaction system is established by optimizing the four main factors that influence the reaction. 2, LAMP real-time turbidity and LAMP staining optimization experiments, on the one hand by means of the new instrument real-time turbidity monitoring loop-mediated isothermal amplification (referred to as real-time turbiditor) LAMP detection of genetically modified soybean reaction Real-time monitoring and analysis; LAMP staining transgenic soybean research and analysis. The final optimized 25μL LAMP reaction system to: magnesium ion concentration was adjusted to 0.2 mmol / L, the amount of Bst DNA polymerase 0.8 μL, of betaine concentration 5 mmol / L, the reaction temperature was 63 ° C. 3, LAMP method with conventional PCR method of comparative analysis, the results show the sensitivity of LAMP is an order of magnitude higher than the PCR, the lowest detection limit was 0.01%. 4 LAMP real-time turbidity method LAMP staining time and sensitivity comparison analysis, nucleic acid dyes can be used to shorten the detection time of 10min-15min, and consistent with the positive rate of turbidity law. 5, in order to shorten the time required for the DNA extraction process detection process, the rapid extraction of soybean seed DNA research, try to soybean dry seeds with proteinase K and SDS method for rapid DNA extraction the LAMP test results show that: proteinase K DNA extraction and the SDS soybean dry seed extraction to meet the needs of LAMP amplification, relative to traditional CTAB method of extracting soybean germ DNA, proteinase K and SDS soybean dry seed extract can save the time of seed germination 10-12 hours.

Studies on a Real-time Turbidimeter-based Loop-mediated Isothermal Amplification for Detection of Roundup Ready Soybean

Abstract

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