Method for rapidly detecting LOX (lipoxygenase) transgenic wheat and kit using method
A fast technology for transgenic wheat, which is applied in the field of molecular genetic breeding, can solve the problems of defective technology and technical inspiration, and has not been applied, and achieve the effects of improved recognition, short detection cycle and low detection cost
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Embodiment 1
[0048] Example 1 LAMP detection kit for LOX transgenic wheat lines
[0049] A test kit using the method for detection, which contains:
[0050] 10×LAMP reaction buffer 2.5 μL, concentration 8 U / μL Bst DNA polymerase 1.0 μL, primers F3 and B3 each at 10 μmol / L concentration 0.5 μL, primers BIP and FIP each at 20 μmol / L concentration 1.6 μL, 1.0 μL of DNA template, 4.0 μL of betaine at a concentration of 5 mol / L, 3.5 μL of dNTPs at a concentration of 10 mmol / L, and MgSO at a concentration of 100 mmol / L 4 Solution 1.0 μL, chromogenic reagent hydroxynaphthol blue 1.0 μL, with ddH 2 O was added to 25 μL; the positive control was 100 μL of transgenic wheat DNA solution with a concentration of 1 μg / μL, and the amount of each positive control was 1 μL; the primers included:
[0051] Outer primer F3: 5'-GTTCAGCCAGTGCTGACAA-3' (SEQ ID No.1);
[0052] Outer primer B3: 5'-AGCCTAGCCAATCTTCACAATC-3' (SEQ ID No.2);
[0053] Internal primer FIP: 5'-CGCCTGCAGGTCGACCATATGGCTGATGCCATCTAG ACT...
Embodiment 2
[0055] Example 2 Simple Detection of LOX Transgenic Wheat and Its Derivatives by LAMP
[0056] The kit described in Example 1 was used for operation.
[0057] Take 1 μL of transgenic wheat DNA to be tested as a template, and prepare a 25 μL reaction system according to the kit requirements:
[0058] Including 2.5 μL of LAMP reaction buffer, 1.6 μL of internal primers FIP and BIP, 0.5 μL of external primers F3 and B3, 4.0 μL of betaine, 3.5 μL of dNTPs, 1.0 μL of hydroxynaphthol blue, MgSO 4 solution 1.0 μL and Bst DNA polymerase 1.0 μL with ddH 2 O make up to 25 μL;
[0059] The mixed reaction was incubated at 63°C for 40-60min, and finally heated at 80°C for 5min to terminate the reaction;
[0060] The reaction solution changes from purple blue to sky blue, indicating that the detected sample is LOX transgenic wheat; otherwise, the sample to be tested is non-LOX transgenic wheat (results such as figure 1 shown).
Embodiment 3
[0061] Example 3 LAMP detection of LOX transgenic wheat and its derivatives
[0062] This embodiment is to verify the detection result of embodiment 2 and carry out repeated tests under the same conditions, but according to the kit described in embodiment 1, except for the chromogen;
[0063] Using the DNA of the sample to be tested as a template, prepare a 25 μL reaction system according to the requirements of the kit. The mixed reaction was incubated at 63°C for 40-60min, and finally heated at 80°C for 5min to terminate the reaction. The reactants show characteristic bands through electrophoresis detection, which proves that the detected sample is transgenic wheat (results such as figure 1 shown), by figure 1 with figure 2 It can be seen that the detection result of the present invention completely corresponds to the gel electrophoresis result, and the accuracy is high.
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