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
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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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