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Molecular detection method for rapid detection of Ile-1781-Leu mutation of anti-fenoxaprop-P-ethyl Beckmannia syzigachne

A technology of molecular detection and lingeria, which is applied in the field of agricultural science to reduce environmental pollution, reduce the probability of false positives, and improve accuracy

Inactive Publication Date: 2014-05-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant reports on the rapid molecular detection of LAMP for the Ile-1781-Leu mutation of the antifenoxaprop-prop-P.

Method used

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  • Molecular detection method for rapid detection of Ile-1781-Leu mutation of anti-fenoxaprop-P-ethyl Beckmannia syzigachne
  • Molecular detection method for rapid detection of Ile-1781-Leu mutation of anti-fenoxaprop-P-ethyl Beckmannia syzigachne
  • Molecular detection method for rapid detection of Ile-1781-Leu mutation of anti-fenoxaprop-P-ethyl Beckmannia syzigachne

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Optimization of LAMP reaction system

[0034] In order to save the detection cost and ensure the stability and reliability of the detection method, the experiment has 2 The concentration (2~6mM), dNTP concentration (0.2~1mM), F3, B3 concentration (0.1~0.8μM), FIP, BIP concentration (0.4~1.2μM) and betaine concentration (0~1.6M) are optimized, The optimal reaction system was determined as follows: Bst DNA polymerase (8U / μL) 0.4μL, 10×ThermoPol1μL, MgCl2 (25mM) 1.8μL, dNTP (10mM) 0.8μL, FIP (20μM) 0.4μL, BIP (20μM) 0.4 μL, F3 (10 μM) 0.6 μL, B3 (10 μM) 0.6 μL, betaine (5M) 1.6 μL, genomic DNA 1.0 μL, dH2O (sterilized distilled water) 1.4 μL.

Embodiment 2

[0035] Example 2 Optimization of LAMP reaction conditions

[0036] In order to obtain the most suitable reaction temperature and time and ensure the efficiency of the detection method, the experiment optimized the reaction temperature (61-65℃) and time (20-100min) in the reaction parameters, and finally determined the most suitable reaction temperature And time were 65℃ and 60min.

Embodiment 3

[0037] Example 3 LAMP reaction sensitivity detection

[0038] In order to determine the lower limit of detection of the LAMP reaction, in this experiment, the genomic DNA extracted and purified by the kit was used as a template in a 10-fold dilution. Using the above-mentioned diluted genomic DNA as a template, LAMP and PCR amplification were performed respectively. figure 2 with image 3 It can be seen that the lowest detection limit of LAMP technology is 5×10 -4 ng / μL, while the lower limit of ordinary PCR detection is only 5×10 -1 ng / μL.

[0039] The detection method established by the present invention can accurately and quickly detect the sperm-resistant fenoxaprop-p-ethyl containing the Ile-1781-Leu mutation, and provides a simple, fast and low-cost method for scientific research and production practice. The detection technology also provides a theoretical basis and technical guidance for the early warning and rational use of the resistant weeds, and has practical and far-re...

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Abstract

The invention discloses a molecular detection method for rapid identification of genotypes of anti-fenoxaprop-P-ethyl Beckmannia syzigachne containing Ile-1781-Leu mutation based on LAMP technology. The detection method can be used for early warning and epidemiological monitoring of Beckmannia syzigachne containing the Ile-1781-Leu mutation. The detection method comprises altogether three main steps: (1) respectively extracting genomic DNA of a sample to be detected; (2) designing specific primers and carrying out LAMP isothermal amplification; and (3) adding the fluorescent dye SYBR Green I into an LAMP amplification product and observing color changes, or carrying out separation through agarose gel electrophoresis with 3.0% agarose and observing amplification results, wherein whether there is a population of Beckmannia syzigachne containing the Ile-1781-Leu mutation is determined based on the results, a population of Beckmannia syzigachne is determined to be a population containing the Ile-1781-Leu mutation if the LAMP amplification product is yellow or electrophoretogram is in the form of a scalariform strip, and a population of Beckmannia syzigachne is determined to be a population not containing the Ile-1781-Leu mutation if the LAMP amplification product is orange or electrophoretogram has no strip amplification. According to the invention, isothermal amplification is realized, expensive instruments and equipment and tedious electrophoresis process are not needed, and detection time and cost are substantially reduced. Compared with common PCR, the detection method provided by the invention has the characteristics of high sensitivity, strong specificity, high detection accuracy, etc.

Description

Technical field [0001] The present invention is a molecular detection method based on loop-mediated isothermal amplification (LAMP) technology for rapid detection of the Ile-1781-Leu mutation of Fenoxoxaprop-p-Pyramid, which belongs to the field of agricultural science and technology. Background technique [0002] Loop-mediated isothermal amplification reaction (LAMP) is a novel isothermal nucleic acid in vitro amplification technology invented by Japanese scholar Notomi et al. in 2000. This method uses a set of (4 types) of specific primers to identify six target genes. Specific areas. The principle of the technology is: under the action of Bst large fragment polymerase, a self-circulating strand displacement reaction is caused. Within 60 minutes of 60-65C, a large amount of target DNA is synthesized while accompanied by a by-product-white magnesium pyrophosphate precipitation. SYBR Green I is a fluorescent dye that shows different colors according to the changes in the composi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/119
Inventor 董立尧潘浪李俊徐洪乐
Owner NANJING AGRICULTURAL UNIVERSITY
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