Aphid aphid specific ss-coi primer, kit containing the primer and detection method thereof
The invention relates to a technology of aphid and a detection method, which are applied in the field of molecular biology and can solve the problems of inability to accurately assess the pest control effect of natural enemies aphids, and achieve the effects of high sensitivity, high accuracy and simple operation.
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Embodiment 1
[0021] Example 1 Amplification Effect of Primers LFSgF / LFSgR on Digemia wheat aphid
[0022] 1) Preparation of the D. wheat aphid genome
[0023] Put the single-headed aphid into a 1.5mL centrifuge tube and grind it, and use the TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit (Spin Column Type) (Tiangen Biological Co., Ltd., Beijing) to extract the single-headed aphid genome . The DNA solution was stored at -20°C for later use, and 1 μL of the solution was taken as a DNA template during PCR amplification.
[0024] (2) Synthesis of specific SS-COI primers for the detection of D. wheat aphids
[0025] The specific SS-COI primer sequence is as follows:
[0026] LFSgF: 5'-CATTATCTAATAATATTGCTCATAAC-3' (SEQ ID No. 1)
[0027] LFSgR: 5'-TGATAAAATTAATAAAATAGCTGTG-3' (SEQ ID No. 2)
[0028] 2) PCR amplification
[0029] The reaction system is 20 μL, including: 10×EasyTaq buffer 2.0 μL, dNTP (2.5 mM) 0.4 μL, EasyTaq DNA polymerase (5U / μL) 0.2 μL, forward and ...
Embodiment 2
[0039] Example 2 Determination of the minimum detectable amount of the primer LFSgF / LFSgR on the aphid
[0040] 1) Preparation of the template genome of the two-handed aphid
[0041] 1) Preparation of the template DNA of the two-handed aphid
[0042] According to Example 1, the genomic DNA of the single-headed aphid was extracted, and then the original template solution was diluted with a 10-fold gradient until no bands were detected, and 1 μL was taken as a template for PCR amplification, which was directly added to the PCR reaction system. The system is the same as that described in Example 1.
[0043] Using primers LFSgF / LFSgR to determine the minimum detection amount, PCR amplification was carried out with different dilutions of D. wheat aphid genomic DNA as a template, and the detection limit of DNA concentration was 0.341 ng / μL ( figure 2 ).
[0044] 2) Preparation of Plasmids of D. aphids
[0045] According to the implementation example 1, the genomic DNA of the ap...
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