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

Inactive Publication Date: 2012-07-04
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional thrips species identification technology is based on the morphological characteristics of adults, and it is difficult to identify immature insects (eggs and larvae), damaged samples, and morphologically very similar species (such as yellow-breasted thrips and rhododendron thrips). It cannot meet the special needs faced in the actual work of agricultural production diagnosis, control and plant quarantine (rapid, accurate, large-scale, high-throughput detection and identification of thrips species)

Method used

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  • Method for assisting in identifying thrips andrewsi and special primer pair thereof
  • Method for assisting in identifying thrips andrewsi and special primer pair thereof
  • Method for assisting in identifying thrips andrewsi and special primer pair thereof

Examples

Experimental program
Comparison scheme
Effect test

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.

...

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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Abstract

The invention discloses a method for assisting in identifying thrips andrewsi and a special primer pair thereof. The specific primer pair is formed by a DNA (Deoxyribonucleic Acid) fragment shown by a sequence 1 of a sequence table and a DNA fragment shown by a sequence 2 of the sequence table. The special primer pair can be used for assisting in identifying the thrips andrewsi. The special primer pair has the following advantages that: (1) the rapid identification for the thrips andrewsi is realized, the identification problem of a non-imago-state sample of the thrips andrewsi is solved, and a new way for identifying two morphological similar thrips (thrips hawaiiensis and the thrips andrewsi) is developed; (2) the operation is simple and convenient, and the method is economic and feasible; the traditional morphological identifying method generally needs a slide sample manufacturing skill and has high requirements for integrity of the sample; but the special primer molecule identifying method can be standardized, the operation is simple and convenient, the needed cost is lower, a staff without a knowledge background of professional taxonomy can conveniently operate and apply the method, and the method is easily popularized and applied in practical work; (3) and the detection sensitivity is high, the elapsed time is short, and the high-throughput detection can be realized.

Description

technical field [0001] The invention relates to the fields of insect quarantine identification and pest monitoring, in particular to a method for assisting the identification of rhododendron thrips and its special primer pair. Background technique [0002] Thrips is a group of harmful organisms, there are about 5500 species, the quarantine and control of thrips is of great economic significance. There are more than 550 species of thrips that have a major impact on the economy, most of which occur in tropical and subtropical regions, causing direct harm to the production of fruits and vegetables, and seriously affecting the import and export trade and transportation of fruit and vegetable products in tropical regions. occupy an important position in. [0003] Thrips are small in size, usually 1-1.5 mm in size, have strong fecundity, like to inhabit hidden places, and are widely distributed. They are important pests in agricultural production and quarantine. Thrips of econom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 伍祎刘奎邱海燕
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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