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A set of primers for identification of stored booklice and its application

A technology for storing booklice and booklice, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection. Easy to operate and use, low cost, accurate and reliable identification results

Active Publication Date: 2019-01-01
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 female adults, which has the disadvantages of requiring high professional skills and taking a long time, especially the accurate identification of non-adult insects, such as eggs, larvae, and pupae, is difficult to achieve, causing quarantine work. very difficult

Method used

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  • A set of primers for identification of stored booklice and its application
  • A set of primers for identification of stored booklice and its application
  • A set of primers for identification of stored booklice and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the design of specific primer pair

[0073] The present invention designs specific primer pairs for eight common stored-grain booklices, as shown in Table 1, on the basis of obtaining the mitochondrial COI gene DNA barcode segments of ten common stored-grain booklices, and through multiple sequence alignment analysis.

[0074] Table 1 List of specific primers for 8 common storage booklice species in the world

[0075]

[0076]

Embodiment 2

[0077] Embodiment 2, the specific detection of primer

[0078] 1. Specific detection of entoF3 and entoR2

[0079] (1) Using the CTAB method to extract the genomic DNA of a single (or multiple) head of the stored booklice from sample 1 to sample 21.

[0080] (2) Use each genomic DNA obtained in step (1) as a template, and use entoF3 and entoR2 as primers to carry out PCR amplification to obtain each PCR amplification product, and simultaneously use ddH 2 O replaced the template as a negative control.

[0081] PCR amplification system (total volume 25 μL): 2×Taq PCR MasterMix (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) 12.5 μL, ddH 2 O 9.5 μL, 1 μL of template with a concentration of 100-300 ng / μL, and 1 μL of primers with a concentration of 10 μM.

[0082] PCR amplification program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 30 sec, 30 cycles; extension at 72°C for 5 min.

[0083]...

Embodiment 3

[0115] Embodiment 3, the sensitivity detection of primer

[0116] 1. Sensitivity detection of entoF3 and entoR2

[0117] (1) Genomic DNA of sample 2 was extracted by CTAB method and diluted to 0.01ng / μL, 0.1ng / μL, 1ng / μL, 5ng / μL, 10ng / μL, 25ng / μL, 50ng / μL, 75ng / μL μL and 100ng / μL concentrations.

[0118] (2) Using the genomic DNA of each dilution obtained in step (1) as a template, and using entoF3 and entoR2 as primers to carry out PCR amplification to obtain each PCR amplification product, and ddH2O instead of the template as a negative control.

[0119] The PCR amplification system is as in Example 2.

[0120] The PCR amplification procedure was as in Example 2.

[0121] (3) Each PCR amplification product is carried out to agarose gel electrophoresis, the result is as follows Figure 9 shown.

[0122] Figure 9 Middle, M: DNA relative molecular weight standard (D2000); 9 different concentrations of DNA in μL and 100ng / μL were PCR amplification products as templates; ...

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Abstract

The invention discloses a primer group for identifying storage book lice and application of the primer group. The primer group comprises at least one primer pair. The primer pairs comprise DNA (deoxyribonucleic acid) molecules which are respectively shown as (1) SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8. The primer group and the application have the advantages that the species of the storage book lice can be quickly and efficiently identified by primers, identification results are accurate and reliable, the primer group is high in detection sensitivity, and high-throughput detection can be implemented; identification objects are free of influence of species gender, developmental stages and integrity degrees of morphological identification characters as compared with the traditional morphological identification methods; identification procedures are easy and convenient to implement and are economical and feasible.

Description

technical field [0001] The invention relates to a group of primers for identifying storage booklice and its application, belonging to the field of biotechnology. Background technique [0002] Booklice, also known as paperlice or ricelice, belongs to the order Psocoptera, the family Liposcelididae, and the genus Liposcelis Motschμlsky 1852. Booklices are tiny insects with a body length of 0.6-1.5 mm and are widely distributed in the world. Most of them live indoors, and a few live outdoors. They are common pests in storage protection. This type of pest has the characteristics of strong activity, wide feeding range, short growth cycle, diverse reproduction methods, carrying a variety of germs, strong resistance, and difficulty in prevention and control. It seriously damages stored items and threatens human health. Booklice pests can spread long distances with stored goods. With the increasing frequency of international trade and stored grain transportation, the risk of the sp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6888
Inventor 李志红杨倩倩
Owner CHINA AGRI UNIV
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