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Specific primer pair for assisting booklice identification and application thereof

A technology of specific primer pairs and assisted identification, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. and other problems, to achieve the effect of simple operation, short time-consuming, improved efficiency and accuracy

Active Publication Date: 2012-03-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional identification of booklice species is mostly based on the morphological characteristics of adults. Due to the small size of booklice, morphological identification requires high professional skills, which is time-consuming and difficult.
Especially for individuals in non-adult developmental stages (such as eggs, larvae, and pupae), morphological identification is difficult to do

Method used

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  • Specific primer pair for assisting booklice identification and application thereof
  • Specific primer pair for assisting booklice identification and application thereof
  • Specific primer pair for assisting booklice identification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the design of specific primer pair

[0058] Based on the mitochondrial cytochrome oxidase subunit I barcode (mtDNA COI barcode) segment of ten common stored grain bookhoppers of the genus Booklice, a specific primer pair for identifying bookhoppers was designed and synthesized:

[0059] Upstream primer (sequence 1 of the sequence listing): 5'-GAACAATAATCGGGAGAGGA-3'; Tm=58.0°C, GC%=45.0;

[0060] Downstream primer (sequence 2 of the sequence listing): 5'-GTAGAAGAATTGCTGTAATAAG-3'; Tm=52.8°C, GC%=31.8.

Embodiment 2

[0061] Embodiment 2, application specific primer is paired to identify bookworm

[0062] The 22 samples were subjected to the following experiments:

[0063] 1. Extract the genomic DNA of a single booklice.

[0064] 2. Using the genomic DNA in step 1 as a template (with water as a negative control), perform PCR amplification with the specific primer pair designed in Example 1 to obtain a PCR amplification product.

[0065] PCR reaction system (26ul): 2×Taq PCR Master Mix 13ul, upstream primer (10uM) 0.5ul, downstream primer (10uM) 0.5ul, genomic DNA 2ul, ddH 2 O 10ul.

[0066] PCR reaction parameters: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 sec, annealing at 50°C for 40 sec, extension at 72°C for 1 min, 32 cycles; 72°C for 8 min; storage at 4°C.

[0067] 3. Perform 1.5% agarose gel electrophoresis on 4 ul of the PCR amplified product from step 2, stain with ethidium bromide (EB), observe in the gel system, and analyze by imaging.

[0068] For th...

Embodiment 3

[0070] Embodiment 3, the sensitivity detection of specific primer pair

[0071] Sample 1 was subjected to the following experiments:

[0072] 1. Extract the genomic DNA of a single booklice.

[0073] 2. Detect the concentration of genomic DNA, and serially dilute it with TE buffer to obtain 8 dilutions; the concentrations of genomic DNA in the 8 dilutions are: 0.1ng / ul, 1ng / ul, 5ng / ul, 10ng / ul, 25ng / ul, 50ng / ul, 75ng / ul and 100ng / ul, dilution 1 to dilution 8 in sequence.

[0074] 3. Using each dilution as a template, perform PCR amplification with the specific primer pair designed in Example 1 to obtain PCR amplification products.

[0075] PCR reaction system (26ul): 2×Taq PCR Master Mix 13ul, upstream primer (10uM) 0.5ul, downstream primer (10uM) 0.5ul, diluent (including genomic DNA) 1ul, ddH 2 O 11ul.

[0076] PCR reaction parameters: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 sec, annealing at 50°C for 40 sec, extension at 72°C for 1 min, 32 c...

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PUM

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Abstract

The invention discloses a specific primer pair for assisting booklice identification and application thereof. The specific primer pair provided by the invention is composed of DNA shown as a sequence 1 in a sequence table and DNA shown as a sequence 2 in the sequence table. The specific primer pair disclosed by the invention can be used for assisting the booklice identification from a molecular level, is free from influences of sample ontogenesis state and sample integrity, and has the advantages of high efficiency, rapidness and accuracy. According to the invention, the booklice variety identification problem which puzzles the stored grain protection and port quarantine departments for a long time is solved. The specific primer pair disclosed by the invention can be widely applied to thegrain storage department and the quarantine department.

Description

technical field [0001] The invention relates to a pair of specific primers for assisting the identification of bookworms and the application thereof. Background technique [0002] Booklice, also known as paper lice and rice lice, belongs to Psocoptera, family Liposcelidiae, and genus Liposcelis, and is a common pest in the protection of stored grains in the world. The pests of the genus Booklice are tiny (adults are about 1mm long) and have strong mobility. They can be transported with stored grains and carried by humans for long distances. They are very easy to establish populations. With the increasing frequency of international trade and stored grain transportation, the risk of the spread of stored grain pests is increasing. The booklice L. corrodens (Heymons) is widely distributed abroad, but has not been reported in my country, and there is a greater risk of invasion. [0003] The genus Liposcelis includes the following species: L. corrodens, L. decolor, L. paeta, L. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 李志红杨倩倩
Owner CHINA AGRI UNIV
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