LAMP primer used for detecting sheep derived material and design method thereof
A design method and sheep-derived technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection. , the effect of high sensitivity
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Embodiment 1
[0055] Example 1 Establishment of the LAMP method for detecting sheep-derived components and optimization of the concentration ratio of internal and external primers
[0056] (1) Take 8 reaction tubes, each with 25 μL of reaction system. Add 15 μL of IMM, 1 μL of F3, 1 μL of B3, and 1 μL of sheep-derived nucleic acid to each tube, add 0.5 μL of FIP and 0.5 μL of BIP to the first reaction tube; add 1 μL of FIP and 1 μL of BIP to the second reaction tube; Add 1.5 μL of FIP and 1.5 μL of BIP to the first reaction tube; add 2 μL of FIP and 2 μL of BIP to the fourth reaction tube; add 2.5 μL of FIP and 2.5 μL of BIP to the fifth reaction tube; add 3 μL of FIP to the sixth reaction tube , BIP 3 μL; add FIP 3.5 μL, BIP 3.5 μL to the seventh reaction tube. Each tube was made up to 25 μL with water. In the eighth reaction tube, water was used instead of sheep-derived component nucleic acid as a blank control.
[0057] (2) Amplify at 65°C for 30 minutes; anneal from 98°C to 80°C at 0...
Embodiment 2
[0059] Example 2 is used to optimize the working concentration of the LAMP method for detecting sheep-derived components
[0060] (1) Take 6 reaction tubes, each with 25 μL of reaction system. Add 15 μL of IMM and 1 μL of sheep-derived nucleic acid to each tube, add 0.5 μL of FIP, 0.5 μL of BIP, 0.25 μL of F3, and 0.25 μL of B3 into the first reaction tube; add 1 μL of FIP, 1 μL of BIP, F3 0.5 μL, B3 0.5 μL; add FIP 1.5 μL, BIP 1.5 μL, F3 0.75 μL, B3 0.75 μL to the third reaction tube; add FIP 2 μL, BIP 2 μL, F3 1 μL, B3 1 μL to the fourth reaction tube; Add 2.5 μL of FIP, 2.5 μL of BIP, 1.25 μL of F3, and 1.25 μL of B3 into the fifth reaction tube; add 3 μL of FIP, 3 μL of BIP, 1.5 μL of F3, and 1.5 μL of B3 into the sixth reaction tube.
[0061] (2) Amplify at 65°C for 30 minutes; anneal from 98°C to 80°C at 0.05°C / s.
[0062] (3) The test results are as follows: figure 2 As shown, S-type amplification curves can appear, and the T of the 1st to 6th reactions M The value...
Embodiment 3
[0063] Example 3 is used to detect the optimization results of LoopF and LoopB of the LAMP method of sheep-derived components
[0064] (1) Take 4 reaction tubes, each with 25 μL of reaction system. Add 15 μL of IMM, 1 μL of sheep-derived component nucleic acid, 2 μL of FIP, 2 μL of BIP, 1 μL of F3, and 1 μL of B3 into each tube; add 1.5 μL of LoopF and 1.5 μL of LoopB to the first reaction tube; add 1 μL of LoopF to the second reaction tube 1 μL of LoopB; add 0.5 μL of LoopF and 0.5 μL of LoopB to the third reaction tube; add 0 μL of LoopF and 0 μL of LoopB to the fourth reaction tube. Replenish with water if the reaction system is less than 25 μL.
[0065] (2) Amplify at 65°C for 30 minutes; anneal from 98°C to 80°C at 0.05°C / s.
[0066] (3) The test results are as follows: image 3 As shown, S-type amplification curves can appear, and the T of the first to fourth reactions M The values (mm:ss) are 7:45, 7:45, 8:45, 18:00 respectively. When LoopF and LoopB ...
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