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Method for rapid identification of liriomya huidobresis blanchard similar species by using PCR-RFLP technology

A technique of PCR-RFLP and Liriomyza, applied in the molecular field of identifying Liriomyza trifolii and its related species, can solve the problems of expensive endonucleases and inability to clearly distinguish them, and achieve the effect of low-cost and rapid detection

Inactive Publication Date: 2008-06-18
CHINA JILIANG UNIV
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to distinguish and identify Liriomyza sativae and Liriomyza sativae which are similar species that are easily confused with its external form, the PCR-RFLP marker technology that exists in the selected internal Dicer is either expensive or cannot clearly distinguish the defects of these three kinds of Liriomyza similaris; a method is proposed to use the appropriate target sequence as the identification basis to clarify the differences between the corresponding sequences of the three common domestic Liriomyza similaris through sequencing , and then select a cheap and stable endonuclease for analysis and identification to achieve a fast, accurate and low-cost method for identifying and distinguishing Liriomyza trifoliata and its related species

Method used

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  • Method for rapid identification of liriomya huidobresis blanchard similar species by using PCR-RFLP technology
  • Method for rapid identification of liriomya huidobresis blanchard similar species by using PCR-RFLP technology

Examples

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Effect test

Embodiment 1

[0041] Example 1: (COII enzyme digestion identification of Liriomyza trifolii, Liriomyza sativae and Liriomyza sativae using DraI)

[0042] Follow these steps:

[0043] 1. DNA extraction and related sequence acquisition:

[0044] Take one adult specimen of Liriomyza trifoliata, Liriomyza sativae and Liriomyza sativae, and extract DNA respectively according to the following steps: Take one specimen and soak it in a 1.5ml centrifuge tube filled with sterile water for 1 hour; take out Grind the specimen in 100 μl extract (Tris: 10 mM, EDTA: 1 mM, SDS: 1%, PH=8.0), add 2.5 μl proteinase K (20 mg / ml) and digest in a water bath at 56 °C for 2 h; add an equal volume of phenol / Extract with chloroform / isoamyl alcohol (25:24:1) mixture, take the supernatant and add 2 times the volume of absolute ethanol to precipitate DNA; finally dissolve DNA with sterile water; store at -20°C as a template for PCR reaction .

[0045] The PCR reaction conditions are as follows: 50 μl of the system,...

Embodiment 2

[0051] Embodiment 2: (Utilize DraI endonuclease to the verification of the homogeneity of different worm states of three kinds of Liriomyza sativae)

[0052] The extraction of DNA from pupae and larvae of Liriomyza trifolii, Liriomyza sativae and larvae of Liriomyza sativae and the acquisition of related sequences are the same as in Example 1; Adults were consistent, therefore, the discrimination analysis using DraI endonuclease will not be disturbed by insect state (Figure 2).

Embodiment 3

[0053] Embodiment 3: (Utilize DraI endonuclease to the verification of different species of Liriomyza pisa and three kinds of Liriomyza sativae)

[0054] Since Liriomyza pisa, which is similar in shape to the three types of Liriomyza sativae, overlaps with Liriomyza sativae on the host, it is necessary to use Liriomyza pisa as a verification species for verification; Same as Example 1), its electrophoresis bands are obviously different from three kinds of Liriomyza sativus enzyme-cut electrophoresis bands, indicating that the identification using DraI endonuclease will not be affected by mixed Liriomyza pisa (Fig. 3).

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Abstract

The invention discloses a method which can rapidly identify liriomyza approximate species by PCR-RFLP technology, which belongs to the insect species identification technical field. The method includes: (1) the DNA extraction of liriomyza trifolii, Americanliriomyza and South American liriomyza samples; (2) obtaining DNA sequence of 805bp by the PCR reaction of COII gene according to insect COII gene universal primers; (3) obtaining the COII sequence respectively through the clone, the transformation and the sequencing; (4) the selection of restriction endonuclease DraI; (5) the distinction of three approximate liriomyzas through a restriction map. The invention has the characteristics of accuracy, stability, fastness, cheapness, etc. And the invention can be applied in related departments of quarantine inspection, forecast, prevention, etc. to realize the rapid detection on the three approximate liriomyzas.

Description

technical field [0001] The invention relates to the technical field of insect species identification, in particular to a molecular method for identifying Liriomyza trifoliata and its close species using PCR-RFLP technology. Background technique [0002] In the genus Liriomyza, there are more than 100 species that can damage or feed on cultivated crops and ornamental crops, of which more than 20 species are of great economic importance. From a global perspective, there are three species of Liriomyza spp. that are most harmful: L. sativae Blanchard, L. huidobrensis Blanchard and L. trifolii Burgess. All three species of Liriomyza sativus have successfully invaded my country, causing huge economic losses to my country's agricultural production; Among them, Liriomyza trifoliata has been identified as a quarantine pest in my country. With the frequent transfer of agricultural products between regions, Its distribution range and degree of harm tend to expand and aggravate. Due to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 商晗武赵永旭崔旭红俞晓平
Owner CHINA JILIANG UNIV
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