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Primer pair for Russian longhorn beetle larva cloning, and applications thereof

A technology of long beetle larvae and primer pairs, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of undescribed larval morphology, lack of larval isolation and identification data, and easily confused identification. , to achieve high specificity and stability

Inactive Publication Date: 2016-04-27
太仓海关综合技术服务中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The taxonomic research of longhorn beetle larvae mainly focuses on the field of individual morphology, but due to the extreme lack of larvae isolation and identification data, many species of longhorn beetle larvae have not been described so far; the same species of longhorn beetle larvae with different geographical distributions have certain differences in morphology; Close species, very similar in shape, easy to be confused when identifying

Method used

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  • Primer pair for Russian longhorn beetle larva cloning, and applications thereof
  • Primer pair for Russian longhorn beetle larva cloning, and applications thereof
  • Primer pair for Russian longhorn beetle larva cloning, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Preparation of the kit.

[0016] 1. Test material

[0017] The longicorn beetle specimens used were the specimens intercepted by the insect laboratories of Jiangsu Entry-Exit Inspection and Quarantine Bureau for many years. The source and collection time of the specimens are shown in Table 1. In addition, 28 beetle COI gene sequences registered in Genbank were selected for comparison, see Table 2.

[0018] Table 1 Name and source of experimental specimens

[0019]

[0020] Table 2 Genbank download sequence information

[0021]

[0022]

[0023] 2. Reagents

[0024] Contains Mg 2＋ 10×PCRBuffer, 2.5mmol / L dNTPs, Taq DNA polymerase was purchased from Dalian Takara Company; DNAMarker and GoldView were purchased from Nanjing Shanggao Biotechnology Company; primers were synthesized by Shanghai GenScript Co., Ltd. Other reagents are imported or domestic analytical reagents.

[0025] 1. Design and synthesis of primers

[0026] According to the compl...

Embodiment 2

[0034] Embodiment 2: identification method

[0035] Follow the steps below:

[0036] (1) Extraction of total genomic DNA: GenMag animal tissue / cell genomic DNA extraction kit: soak the worm body in alcohol, select muscle tissue of appropriate size, wash 2-3 times with sterile water, drain the water, and The tissue was ground, and an appropriate amount of lysate and proteinase K was added, and the tissue was warmed at 55°C to completely lyse the tissue. Then add magnetic beads and buffer to make the DNA adsorb to the magnetic beads, use WashBuffer to remove impurities, after the removal of impurities, add a small amount of ElutionBuffer to dissolve the DNA to obtain a genomic DNA solution, and store at -20°C.

[0037] (3) PCR amplification: The PCR reaction system uses a 50 μl standard reaction system, which contains 10×PCRBuffer (Mg 2＋ ) 5μl, dNTPs (2.5mmol / L) 4μl, Taq DNA polymerase 0.4 μl, cDNA template 2 μl, upstream and downstream primers 1 μl each, add ddH 2 O to make...

Embodiment 3

[0044] Embodiment 3: specificity experiment

[0045] Using the same method as in Example 2, the DNA of 28 kinds of beetles was extracted and used as a template to carry out PCR amplification with the designed primers. Using 1% agarose for gel electrophoresis, only a 481bp fragment was amplified from Russian longhorn beetle larvae (see figure 1 ), sequenced the PCR product of the pigeon fine ring positive disease material, compared it with the published sequence in GenBank, and applied the Mega5.0 software to analyze the COI gene sequence of longhorn beetle, and found that the conserved site was among the 481 sites 90, 391 mutation sites, 375 informative sites, and 16 self-descended sites. Considering the high degree of heterogeneity among different species of Russian longhorn beetle larvae, it can be shown that the amplified fragment is the Russian longhorn beetle larvae COI gene, while none of the other 28 species of longhorn beetles amplified fragments or bands, which indi...

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Abstract

The present invention discloses a primer pair for Russian longhorn beetle larva COI gene cloning, wherein the nucleotide sequences of the primer pair are represented by SEQ ID NO:1 and SEQ ID NO:2. The present invention further discloses an identification kit for Russian longhorn beetle larva. According to the present invention, with the primer pair, the nucleotide sequence of the Russian longhorn beetle larva COI gene can be successfully amplified, the cloning conditions and the cloning system are optimized to prepare the identification kit for the Russian longhorn beetle larva, the kit has characteristics of high specificity and high stability, the sensitivity achieves 5 pg, and the Russian longhorn beetle larva can be quickly and accurately identified, such that the kit can be adopted as the effective tool for the rapid Russian longhorn beetle larva identification.

Description

technical field [0001] The invention relates to the field of classification and identification of cervix larvae, in particular to a pair of primers for cloning Russian longhorn larvae and an application thereof. Background technique [0002] The taxonomic research of longhorn beetle larvae mainly focuses on the field of individual morphology, but due to the extreme lack of larvae isolation and identification data, many species of longhorn beetle larvae have not been described so far; the same species of longhorn beetle larvae with different geographical distributions have certain differences in morphology; Close species, very similar in shape, easy to confuse identification. Therefore, along with the development of molecular biology, nucleic acid sequence analysis (DNA sequence analysis) is used more and more in the molecular identification of longhorn beetle larvae, which is of great significance in entry-exit inspection and quarantine work. [0003] Mitochondrial DNA (mtD...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 顾忠盈吕飞
Owner 太仓海关综合技术服务中心
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