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LAMP (Loop-Mediated Isothermal Amplification) rapid detection method and application of glyphosate-resist transgenic soybean

A technology of genetically modified soybeans and detection methods, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of not providing LAMP detection pictures, etc., achieving simple operation, high sensitivity, and low equipment requirements. Effect

Inactive Publication Date: 2014-03-19
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, strictly speaking, although the name of the article by Lan Qingkuo et al. is called LAMP detection, it is not actually LAMP detection. The paper does not provide any pictures of LAMP detection.

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) rapid detection method and application of glyphosate-resist transgenic soybean
  • LAMP (Loop-Mediated Isothermal Amplification) rapid detection method and application of glyphosate-resist transgenic soybean
  • LAMP (Loop-Mediated Isothermal Amplification) rapid detection method and application of glyphosate-resist transgenic soybean

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 PCR amplification of the endogenous gene LECTIN of glyphosate-resistant transgenic soybean and the specific exogenous gene CP4-EPSPS.

[0061] The amplification primers of endogenous gene LECTIN refer to GB / T19495.4-2004, and the amplification primers of exogenous specific gene CP4-EPSPS refer to the method of Sun Hongwei et al. Qualitative PCR detection [J]. Food Science, 2008,29(2):234-237.), detailed primer sequences are shown in Table 1.

[0062] Table 1 PCR amplification primer sequence

[0063]

[0064] One-step double PCR was used to amplify, the PCR system used was 25 μl, 10×Buffer (containing Mg2+) 2.5 μl, 10 mM dNTP 2 μl, primers Lectin-F, Lectin-R, Cp4-epsps-F and Cp4-epsps-R (10 μmol / L) 1 μl each, Taq enzyme (5U / μl, Takara) 0.5 μl, template DNA 1 μl, sterilized ddH 2 O to make up to 25 μl. PCR amplification conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C fo...

Embodiment 2

[0065] Example 2 Establishment of the method for detecting glyphosate-resistant transgenic soybeans by LAMP technology

[0066] 2.1 Design of primers for glyphosate-resistant transgenic soybean LAMP

[0067] According to the CP4-EPSPS gene sequence, design and screen the following LAMP primers and probes (such as figure 2 shown), the primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd. The sequence is as follows:

[0068] ①F3: 5'-TCCTCGACGGCCTTCC-3';

[0069] ②B3: 5'-GCACCGTGACGCCCTT-3';

[0070] ③BIP: 5'-CGAAGTCATCAACCCGCGCCAGCGTGGAGGAGCGAAC-3';

[0071] ④ FIP: 5'-TGGGGTTCATCAGCACGTTGAGTTGCGGCCCTGCTTGT-3';

[0072] ⑤LB: 5'-GAAGACGTGGCGGACCT-3';

[0073] ⑥LF: 5'-ATGGTGACGTCGGAGCC-3'

[0074] 2.2 LAMP reaction system configuration: outer primers F3 and B3 each 0.2 μmol / L, inner primers FIP and BIP each 1.6 μmol / L, loop primers LB and LF each 0.4 μmol / L, 10mmol / L dNTP, 20mmol / L Tris-HCl (pH8.8), 10mmol / L KCl, 5mmol / L MgSO 4 , 10mmol / L (NH 4...

Embodiment 3

[0078] Example 3 Detection of the reaction time of the ring-adding primer

[0079] The DNA used as J9331 in soybean was used as a template, and LAMP detection was performed with and without loop primers respectively, and the reaction conditions were the same. Set six reaction times of 15, 30, 45, 60, 75, and 90 minutes for LAMP reaction. After the reaction, add 2 μL of prepared SBRY Green I to the reactant, mix well, and observe the color change. Such as Figure 4 As shown in A, the loop-adding primer LAMP reaction amplifies within 45 minutes, and the color turns green, as shown in Figure 4 As shown in C, without the addition of loop primers, LAMP detection does not amplify until 75 minutes, and the color turns green. Take 2μl of the product and electrophoresis on 2% agarose gel, stain with EB, observe and take pictures under ultraviolet light. Figure 4 As shown in B, it can be observed that the LAMP reaction with loop primers appears a ladder-shaped band at 45 min, whil...

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Abstract

The invention relates to an LAMP (Loop-Mediated Isothermal Amplification) rapid detection method and application of a glyphosate-resist transgenic soybean. The method comprises the following steps: designing and selecting 6 LAMP primers F3, B3, FIP, BIP, LB and LF according to the specificity of a CP4-EPSPS gene sequence; and preparing and optimizing an LAMP reaction system. The glyphosate-resist transgenic soybean can be rapidly detected through DNA extraction of the glyphosate-resist transgenic soybean, loop-mediated isothermal amplification, amplification output color development and observation. According to the detection method, the specificity is strong, the sensitivity is high, the detection is rapid, the cost is low, the operation is convenient, and application values in rapid detection of transgenic soybean and processed goods and field monitoring of gene flow of the transgenic soybean are high.

Description

technical field [0001] The invention relates to a rapid detection method and application of glyphosate-resistant transgenic soybean LAMP, belonging to the field of biotechnology. Background technique [0002] Since the first genetically modified tobacco came out in 1983, research on genetically modified crops has developed rapidly and has a history of 30 years. According to the 2012 report of the International Agricultural Biotechnology Industry Application Service Center (ISAAA), the planting area of ​​genetically modified crops increased by 100 times in the 17 years from 1996 to 2012, from 1.7 million hectares to 170 million hectares. In 2012, the planting area of ​​genetically modified crops in developing countries accounted for 52% of the world, surpassing the planting area of ​​genetically modified crops in developed countries (48%). The top five developing countries planting genetically modified crops are China, India, Brazil, Argentina and South Africa (Clive James, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/119
Inventor 彭德良朱琳峰黄文坤彭焕孔令安贺文婷
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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