A specific primer for detecting pine needle sheath gall midge and its application
A specific and sheath gall technology is applied in the field of specific primers for detecting pine needle sheath gall mosquitoes, which can solve the problems of time-consuming, lack of identification features, laborious and other problems, and achieve the effect of high specificity and high sensitivity
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Embodiment 1
[0023] The extraction of embodiment 1 insect genome DNA
[0024] Genomic DNA collected from different species of Ephididae collected from 4 regions in China was extracted by DNA micro-extraction kit.
[0025] (1) Put the whole worm body into a 1.5ml centrifuge tube, add 200ul buffer PBS, and grind thoroughly with a grinding rod.
[0026] (2) After adding 150ul buffer PBS and 0.9ul Rnase A, grind gently for 30s.
[0027] (3) Collect 350ul of ground tissue homogenate and transfer it to a 2ml centrifuge tube. If the homogenate volume is less than 350ul, add PBS to 350ul.
[0028] (4) Add 150ul buffer C-L and 20ul proteinase K, immediately vortex and shake for 1min to mix evenly, after a brief centrifugation, put the centrifuge tube in a 56°C water bath for 10min.
[0029] (5) Add 350ul buffer P-D, vortex for 30s to mix evenly, and centrifuge at 12000×g for 10min.
[0030] (6) Place the DNA preparation tube in a 2ml centrifuge tube, transfer the mixture in the previous step to ...
Embodiment 2
[0036] Example 2 Amplification of the Gall Mosquito COI Gene Sequence
[0037] According to literature reports, the general primers for synthesizing COI gene sequence amplification of gall midge insects are as follows:
[0038] YWJ: 5'-AATTGGWGGWTTYGGAAAYTG-3'
[0039]YWN: 5'-GCTCGAGTATCAACGTCTATWCC-3'
[0040] PCR amplification reaction using GreenMaster Mix Kit (Promega), the total system is 25 μl, and the components are shown in Table 1:
[0041] Table 1 PCR reaction system
[0042]
[0043] After mixing evenly, place it in a PCR machine for amplification. The PCR reaction program is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and a total of 30 cycles; Extend at ℃ for 5 minutes and store at 4℃. After the completion of the amplification, the amplified product was detected by 1.5% agarose gel electrophoresis (voltage 120V, time 25 minutes, 1×TAE as the electrophoresis ...
Embodiment 3
[0044] Example 3 Multiple Sequence Alignment of Related Species and Design of Species-Specific Primers
[0045] The PCR product was sent to Nuosai Biological (Beijing) Co., Ltd. for bidirectional sequencing to obtain the COI sequence. Sequences were spliced using DNAstar, redundant sequences were removed, and the sequence results were compared by Blast sequence in GeneBank. Based on the comparative analysis of the sequencing results of the four species of gall midges and the base sequences of the other four species of gall midges published in the database, a pair of SS-COⅠ primers (TJSSF1 / TJSSR1) specific to the gall midges were designed using the software PrimerPrimer 5.0 :
[0046] TJSSF1:5'-CAGGTAAAGAAAAGTAAAAGTAGAATTGTTGTAATT-3';
[0047] TJSSR1: 5'-GATTTTGATTACTTCCCCCCCTCTATTTC-3'.
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