Specific primer pair for assisting booklice identification and application thereof

A specific primer pair and auxiliary identification technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of high technical requirements for morphological identification, difficult and difficult morphological identification and other problems, to achieve the effect of simple operation, short time consumption, improved efficiency and accuracy

Active Publication Date: 2013-05-01
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] Example 1. Design of specific primer pairs

[0058] Based on the mitochondrial cytochrome oxidase subunit I barcode (mtDNA COI barcode) segment of ten common stored-grain booklices of the genus Booklice, we designed and synthesized specific primer pairs that can identify booklice:

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

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

Embodiment 2

[0061] Example 2. Application of specific primer pairs to identify book lice

[0062] Perform the following experiments on 22 samples:

[0063] 1. Extract the genomic DNA of single-headed book lice.

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

[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: 95°C pre-denaturation 3min; 95°C denaturation 30sec, 50°C annealing 40sec, 72°C extension 1min, 32 cycles; 72°C 8min; 4°C storage.

[0067] 3. Carry out 4ul of the PCR amplification product of step 2 on 1.5% agarose gel electrophoresis, stain with ethidium bromide (EB), observe and image analysis in the gel system.

[0068] See the electrophoresis results of each sample figure 1 . figure 1 In, M: DNA...

Embodiment 3

[0070] Example 3. Sensitivity detection of specific primer pairs

[0071] Experiment with sample 1 as follows:

[0072] 1. Extract the genomic DNA of single-headed book lice.

[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, which are diluent 1 to diluent 8.

[0074] 3. Using each dilution as a template, PCR amplification was performed 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 (containing genomic DNA) 1ul, ddH 2 O 11ul.

[0076] PCR reaction parameters: 95°C pre-denaturation 3min; 95°C denaturation 30sec, 50°C annealing 40sec, 72°C extension 1min, 32 cycles; 72°C 8min; 4°C storage.

[0077] 4. Perform 4ul of the PCR...

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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 specific primer pair for assisting the identification of book lice and its application. Background technique [0002] Booklice, also known as paper lice and rice lice, belongs to the order Psocoptera, Liposcelidiae, and Liposcelis, and is a common pest in the protection of grain storage in the world. Booklice pests are tiny (the adult body length is about 1mm), and they have strong mobility. They can be transported with stored grains and carried by humans. They are easy to establish populations. They are highly resistant and difficult to control, which seriously endangers the safety of stored grains. With the increasing frequency of international trade and storage of grain, the risk of spread of stored grain pests is increasing. L. corrodens (Heymons) is widely distributed abroad, but it has not been reported in my country, and there is a greater risk of invasion. [0003] Liposcelis includes the following species: L.corrodens, L....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李志红杨倩倩
Owner CHINA AGRI UNIV
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