A primer pair used for identifying grape blossom winglet moths and application thereof

A technology of grape flower and small curly moth, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of difficult identification, low accuracy of identification results, and low applicability, etc. Achieve the effect of shortening customs clearance time, high sample integrity and strong practicability

Active Publication Date: 2019-01-11
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the above-mentioned problems that the identification method of the grape moth is morphological identification, which requires high sample quality and professional knowledge of the identification personnel, low applicability, high identification difficulty, and low accuracy of identification results, the present invention provides grape A pair of specific primers for Toricula variegata, and the use of the primer pair to identify Toricula vinifera, the method has the characteristics of rapidity, accurate identification results, easy operation, high sensitivity and strong specificity, etc.

Method used

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  • A primer pair used for identifying grape blossom winglet moths and application thereof
  • A primer pair used for identifying grape blossom winglet moths and application thereof
  • A primer pair used for identifying grape blossom winglet moths and application thereof

Examples

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Effect test

Embodiment 1

[0020] Embodiment 1 is used to identify the specific PCR primer sequence design of vine toricula

[0021] First, find the full-length mitochondrial COII gene sequences of the vine toricula and 10 reported Lepidopteran toricidae and their morphologically similar species on NCBI;

[0022] Secondly, using MEGA6.0 to compare the COII gene sequences of the grape moth and 10 morphologically similar species, so as to determine the unique COII gene fragment of the grape moth;

[0023] Finally, Primer5 was used to design a pair of specific primers for the specific COII gene fragment of Toricula vine.

[0024] Primers were designed according to the specific sequences obtained above, and the primer sequences were:

[0025] The nucleotide sequence of the upstream primer is as described in SEQ ID NO:1, namely: 5'-CAAAATAGAGCCTCCCCT-3'; the nucleotide sequence of the downstream primer is as described in SEQ ID NO:2, namely: 5'-AAACGACCTGGATTAGCAT-3' .

Embodiment 2

[0026] Example 2PCR primer sequence is used to identify the grape tortrix

[0027] 1. Collection of Insect Samples

[0028] From March to May 2018, the larvae of the grape tortilla moth were collected in Chile and Argentina, and the larvae and adults of the grape tortilla moth were collected in Israel and Italy. After accurate morphological identification by experts, they were placed in absolute ethanol and stored in - 20°C.

[0029] Table 1 The collected moth sample information of the present invention

[0030]

[0031] 2. Genomic DNA Extraction

[0032] Dilute the collected samples with ethanol with a concentration gradient of 75%, 50%, 30%, 10%, and 0%, each concentration for 2 minutes; take it out from the sterile water, and use filter paper to dry the water on the insect body; Place in a sterile 1.5ml centrifuge tube; after grinding thoroughly with a grinding rod, add 100μl LB 2 and 20μl Proteinase K (to ensure that the tissue is completely in the centrifuge tube);...

Embodiment 3

[0042] Example 3 Sequence Alignment of Toricula vine and other similar species

[0043] 1. Download the COII gene sequences of the reported species of Lepidoptera Toricidae and the similar species of Toricula vinifera from NCBI.

[0044] 2. Using the specific primers provided by the present invention to amplify and sequence the COII gene sequence of Toricula vine and the COII gene sequences of other similar species are compared by software CLUSTALX1.83.

[0045] 3. Experimental results

[0046] Through sequence comparison, we found that the similarity between the COII gene sequence of the vine toricula obtained by the amplification and sequencing of the specific primers provided by the present invention and the COII gene sequence of the vine toricula downloaded from NCBI is 100%, The similarities between the remaining 10 and its close species and the COII gene sequence of the vine toricella were all below 92%, which indicated that the primers provided by the present invention...

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Abstract

The present invention provides a primer pair used for identifying grape blossom winglet moths, the upstream primer nucleotide sequence of which is shown as SEQ ID NO: 1, namely: 5 '-CAAAATAGAGCCTCCCCT-3'; A nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, i. E., 5 '-AAACGACCTGGATTAGCAT-3. The invention provides the application of the primer pair. The invention provides the specific primer pair of the grape blossom winglet moth and the method for identifying the grape blossom winglet moth by using the primer pair. The method has the characteristic of fast, accurate identification result, easy operation, high sensitivity and strong specificity.

Description

technical field [0001] The invention belongs to the technical field of molecular identification, and in particular relates to a pair of primers for identifying a quarantine pest, Toricula vine, and a molecular identification method for Toricula vine using the primers. Background technique [0002] Lobesia botrana (Lobesia botrana) belongs to Lepidoptera (Lepidoptera), Tortricidae (Tortricidae), Lobesia (Lobesia), is a worldwide pest that seriously damages the flowers and fruits of various economic plants, In many countries in the world including my country, it is listed as an important entry plant quarantine pest insect. The origin of the worm is Italy, and it has now spread to all of Europe, northern and western Africa, the Middle East and Far East of Asia, and parts of the United States. At present, there are no reports of this species occurring in my country. [0003] The insect has one to several generations a year, and the specific number of generations is related to fa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 苏莎陈茂华王雪婷彭雄田锐铮
Owner NORTHWEST A & F UNIV
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