Specific identification primers and identification methods for the farmland spiders Octopus ornate and Octopus coniferina
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A technology of octopus cone-bellied and specific primers is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The effect of overcoming difficulties and shortening the cycle
Active Publication Date: 2016-08-24
CHINA JILIANG UNIV
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However, there are no reports at home and abroad on the use of molecular markers to identify farmland spiders
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Embodiment 1
[0033] Example 1: Design of specific primers for octopus octopus and octopus ornate and rapid identification method thereof
[0034] (1) Specimen collection and identification and total genome DNA extraction
[0035] The octopus species used in the examples were collected from the farmland of Yuyao City, Zhejiang Province by trap method. The collected specimens were identified with a stereo microscope to determine the species, and the spider specimens used for molecular biology experiments were soaked in 100% alcohol and stored in a 4°C refrigerator. DNA extraction kits were used to extract the total genomic DNA of Osmanthus orientalis. To avoid contamination, the head, chest and abdomen were discarded, and DNA was extracted only from the legs. DNA extraction was directly performed using a DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). The extracted total genome was stored at –20°C for future use.
[0036] (2) PCR amplification and sequence determination of...
Embodiment 2
[0049] Example 2: Using specific primers to quickly identify octopus octopus and octopus ornate
[0050] (1) Primer specificity detection
[0051] In order to detect the specificity of the primers designed under conventional PCR conditions, in addition to the cone-bellied octopus and ornate octopus to be identified and differentiated, the experimental materials also included the anterior-toothed octopus, lin-patterned octopus, black octopus and long claw The octopus spiders with higher homology among the 4 octopus spiders such as octopus carried out PCR reaction together.
[0052] The PCR reaction system is 50μl, including: 2μl template DNA (about 20ng), 5μl 10×PCR buffer, 4μl 2.5mM MgCl 2 , 1 μl 10 mM dNTP, 2 μl 10 mM mixed primers (COI-F, Tm-R and Tn-R), 0.5 μl 5U / μl Taq DNA polymerase, add deionized water to adjust to a final volume of 50 μl;
[0053] The PCR reaction conditions were: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing a...
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Abstract
The invention discloses a specificity identification primer for farmland spider tetragnatha nitens audouin and T.maxillosa thoren and a method for rapidly identifying the two farmland tetragnatha spiders. The method for rapidly identifying the two farmland tetragnatha spiders comprises the following steps: firstly, extracting spider genome total DNA, and carrying out amplification on the two tetragnatha spiders by utilizing a general primer for a cytochrome oxidase i (COI) gene while the extracted spider genome total DNA is taken as a PCR (polymerase chain reaction) template; secondly, designing a species-specific primer according to sequence difference, mixing with a primer of the general primer to form a mixed primer, then carrying out specificity PCR amplification while the spider genome total DNA is taken as a template, carrying out agarose gel electrophoresis detection on an amplification product, and determining species of the spiders according to fragment length. The method for identifying the two farmland tetragnatha spiders has the characteristics of speediness, high efficiency and convenience, and requirement on professional knowledge of an appraiser is low; meanwhile, precise identification can be carried out on an incomplete individual, so that the method for identifying the two farmland tetragnatha spiders is more accurate and reliable compared with the traditional form-depended characteristic identification.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a specific identification primer and identification method for farmland spider octopus orchids and octopus coniferina. Background technique [0002] Farmland spiders are important predatory natural enemies of pests and play an important role in the process of integrated pest management in farmland. Protecting the natural enemy species pool and promoting the reconstruction of the natural enemy community can effectively control the occurrence and damage of many agricultural pests, such as planthoppers, leafhoppers, borers, cotton aphids and other pests. The octopus spider belongs to the order Aranea, the suborder Arachne, and the superfamily Arachnioidae, and is the dominant species among farmland spiders. However, the appearance and morphology of the spiders of the genus Octopus are very similar (the egg stage is even more difficult to distinguish), and the identification...
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