LAMP detecting primer group, kit and detecting method for cry3A gene in transgenic plant
A technology of transgenic plants and detection kits, which is applied in the field of molecular biology, can solve the problems such as the detection methods and kits for cry3A gene in transgenic plants, etc., and achieves the effects of simple operation, high sensitivity and strong specificity.
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Embodiment 1
[0044] Example 1 Kit without chromogen and detection method thereof:
[0045] Prepared according to the following recipe cry3A Gene LAMP Detection Kit:
[0046] (1) Detection primer solution: Synthesize outer primer 1, outer primer 2, inner primer 1, and inner primer 2, make primer dry powder into mother solutions with a concentration of 100 μmol / L with sterilized deionized water, and then take 7.5 μL outer primer Primer 1, 7.5 μL outer primer 2, 60 μL inner primer 1, 60 μL inner primer 2, 15 μL sterilized deionized water, mix thoroughly to make 150 μL cry3A Gene LAMP detection primer solution, wherein the primer sequences are:
[0047] Outer primer 1: AAGCCCCACCTGTTCGA (SEQ ID NO: 1);
[0048] Outer primer 2: GGCTCGCTGCTCTTGTTG (SEQ ID NO: 2);
[0049] Internal primer 1: AGTTGAAGCTGTCGTTGCCGTACCTGCACCGCATCCAGTTC (SEQ ID NO: 3);
[0050] Inner primer 2: GGAGCGGCAACTACGTGAGCAGAAGGGGCTGGTGATGA (SEQ ID NO: 4);
[0051] (2) Loop primer solution: To synthesize loop primers, p...
Embodiment 2
[0065] Example 2 Kit containing chromogen and detection method thereof
[0066] Prepared according to the following recipe cry3A Gene LAMP Detection Kit:
[0067] (1) Detection primer solution: Synthesize outer primer 1, outer primer 2, inner primer 1, and inner primer 2, make primer dry powder into mother solutions with a concentration of 100 μmol / L with sterilized deionized water, and then take 7.5 μL outer primer Primer 1, 7.5 μL outer primer 2, 60 μL inner primer 1, 60 μL inner primer 2, 15 μL sterilized deionized water, mix thoroughly to make 150 μL cry3A Gene LAMP detection primer solution, wherein the primer sequences are:
[0068] Outer primer 1: AAGCCCCACCTGTTCGA (SEQ ID NO: 1);
[0069] Outer primer 2: GGCTCGCTGCTCTTGTTG (SEQ ID NO: 2);
[0070] Internal primer 1: AGTTGAAGCTGTCGTTGCCGTACCTGCACCGCATCCAGTTC (SEQ ID NO: 3);
[0071] Inner primer 2: GGAGCGGCAACTACGTGAGCAGAAGGGGCTGGTGATGA (SEQ ID NO: 4);
[0072] (2) Loop primer solution: To synthesize loop primers,...
Embodiment 3
[0087] Embodiment 3 Specificity experiment of kit and detection method
[0088] Experiments were carried out on the specificity of the kit described in Example 2 and its detection method, and the test samples included: transgenic corn MIR604 ( cry3A ), MON89034 ( cry1A.105 , cry2Ab ), MON810 ( cry1Ab ), Bt11 ( cry1Ab ), Bt176 ( cry1Ab ), MON863 ( cry3Bb ), MON88017 ( cry3Bb ), TC1507 ( cry1F ), 59122 ( cry34Ab , cry35Ab ), transgenic cotton MON531 ( cry1Ac ), MON15985 ( cry1Ac , cry2Ab ), transgenic rice KF-6 ( cry1Ab ), TT51-1 ( cry1Ab / Ac ) and other 13 insect-resistant transgenic crops, as well as non-transgenic corn.
[0089] In this example, only the cry3A The transgenic corn MIR604 sample reaction tube of gene appears green, shows that kit of the present invention and detection method are right to cry3A Genes are very specific.
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