Method for assisting in identifying thrips andrewsi and special primer pair thereof
A technology for assisting identification and rhododendron thrips, which is applied in the field of insect quarantine identification and pest monitoring, can solve the problems of identification of species that are very similar in form and cannot be satisfied, and achieves easy operation and application, short time-consuming and simple operation. Effect
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Embodiment 1
[0019] Embodiment 1, the design of specific primer pair
[0020] Start by collecting thrips species that are commonly found on fruits, vegetables and horticultural plants, including the morphologically similar thrips yellow-breasted and rhododendron thrips. The mitochondrial cytochrome oxidase subunit I barcode (mtDNA COI barcode) sequence was obtained by genomic DNA extraction and PCR amplification, and the mtDNA COI barcode gene sequences of other species of Thrips Linnaeus in the BOLD database and GenBank were collected. On this basis, the DNAMAN software was used to compare and analyze the mtDNA COI barcodes of different species to find the specific sequence segment of Rhododendron thrips. Specific primer pairs of Rhododendron cuckoo were artificially designed, and the primers were evaluated and checked with Oligo software and Blast program. Under the standard PCR reaction system and reaction conditions, the specific primers are screened, and only the target species corre...
Embodiment 2
[0024] The specificity of the primer pair designed by embodiment 2, embodiment 1
[0025] Rhododendron thrips (Thrips andrewsi), yellow-breasted thrips (Thrips hawaiiensis) and common large thrips (Megalurothrips usitatus): The public can obtain them from the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. The literature mentioning the above species is: Zhang Guiling . Taxonomy of Thrips in China: [Master's Dissertation]. Shaanxi: Northwest Agriculture and Forestry University, 2003.
[0026] Thrips parvispinus: The public can obtain it from the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. The literature mentioning this species is: Mound, L.A.&Collins D.W., (2000) A south east Asian pest species newly recorded from Europe: Thrips parvispinus (Thysanoptera : Thripidae), its confused identity and potential quarantine significance. Journal of European Entomology, 97, 197-200.
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Embodiment 3
[0041] The sensitivity of the primer pair designed by embodiment 3, embodiment 1
[0042] 1. Extract the genomic DNA of sample 1 in Table 1.
[0043] 2. Serially dilute the genomic DNA extracted in step 1 with TE buffer to obtain 8 dilutions; the concentrations of genomic DNA in dilution 1 to dilution 8 are: 100ng / μL, 50ng / μL, 25ng / μL, 10ng / μL, 1ng / μL, 0.1ng / μL, 0.01ng / μL, 0.001ng / μL.
[0044] 3. Using each dilution as a template (water as a negative control), carry out PCR amplification with the specific primer pair designed in Example 1 to obtain a PCR amplification product.
[0045] The reaction system of PCR amplification and the cycle parameters of PCR amplification are the same as those in Example 2.
[0046] 4. Take 5 μL of the amplified product for 1.5% agarose gel electrophoresis, observe and image analysis in the gel system.
[0047] see results figure 2 , Lane M corresponds to the DNA relative molecular weight standard, lanes 1-8 correspond to dilution 1 to dil...
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