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Primer pair and kit for identifying or assisting identification of stored bean weevil

A technology for auxiliary identification and primer pairs, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of heavy workload, achieve short time-consuming, simple operation process, and general-purpose technology strong effect

Active Publication Date: 2016-08-24
INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The result of restriction fragment length polymorphism PCR technology is relatively accurate, but the workload of screening the required restriction endonucleases is relatively large
[0006] At present, there is no efficient and convenient identification method for identifying different types of bean weeds in storage

Method used

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  • Primer pair and kit for identifying or assisting identification of stored bean weevil
  • Primer pair and kit for identifying or assisting identification of stored bean weevil
  • Primer pair and kit for identifying or assisting identification of stored bean weevil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The design of embodiment 1 specific primer pair

[0059] By searching and querying the mtDNA CO Ⅰ sequences of Pimus chinensis, C. spp., Vicia bean, and Mung bean in GenBank, the mtDNA CO Ⅰ sequences of the target species were compared and analyzed with DNAMAN software. According to the analysis results, the target gene sequence Specific primers were manually designed, and the primers were evaluated by Oligo software, and the Tm value, GC%, mismatch, dimer and hairpin structure of the primers were checked, and the homologous sequences were checked by the Blast program in GenBank.

[0060] The 5 pairs of primers designed are shown in Table 1:

[0061] Table 1 is the specific primer sequence

[0062]

Embodiment 2

[0063] The identification of embodiment 2 bean weevil

[0064] 1. Extraction of soybean elephant DNA

[0065] Genomic DNA was extracted by salting-out method from single-headed Elephant, including Elephus piscifolia, Elephus four-striped, Elephus faba, Elephus beanus and Elephus mung bean. The above species of bean weeds were all confirmed by morphological characteristics.

[0066] Grind it into a powder in a 1.5ml centrifuge tube with liquid nitrogen, and quickly add 300μL TNES (50mM Tris, pH7.5, 400mM NaCl, 20mM EDTA, 0.5% SDS) and 100μg / mL proteinase K, in Incubate at 37°C for 4 h; add 85 μL of 5M NaCl, shake vigorously for 15 s, and centrifuge at 14,000 rpm for 5 min; take the supernatant, add an equal volume of ice-cold absolute ethanol, and mix gently; centrifuge at 14,000 rpm for 5 min, and wash once with 70% ethanol. The precipitate was dried at room temperature, and an appropriate amount of 50 μL sterile water was added to dissolve the extracted DNA. Then, they wer...

Embodiment 3

[0078] Sensitivity detection of embodiment 3 specific primer pairs

[0079] The numbering obtained in embodiment 2 is carried out serial dilution with distilled water respectively to obtain in the embodiment 2 and be numbered 1-5 and be the picamophora, four-striped bean elephant, broad bean elephant, common bean elephant and mung bean elephant genomic DNA to carry out serial dilution with distilled water, obtain 5 kinds of dilutions; Genomic DNA concentrations were: 50ng / μl, 25ng / μl, 10ng / μl, 5ng / μl, 1ng / μl, 0.5ng / μl, 0.1ng / μl, 0.05ng / μl. Each dilution was used as a template (water was used as a negative control), and the above-mentioned 5 pairs of specific primers were used for PCR amplification of its specific species.

[0080] The PCR reaction system is as follows (25 μL): 10×buffer (containing Mg 2+ ) 2.5 μL, 2.5 mM dNTP 2.5 μL, 10 μM primers (upper and downstream primers) 1.0 μL each, 2.5 U / μL Taq enzyme 0.2 μL, genomic DNA 1 μL, ddH 2 O 16.8 μL.

[0081] PCR reaction...

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Abstract

The invention discloses a pair of primers for identifying or assisting in identifying a stored bean elephant and a kit thereof. The primer pair provided by the present invention consists of the following 5 pairs of primer pairs: primer pair 1 (sequence 1 and sequence 2), primer pair 2 (sequence 3 and sequence 4), primer pair 3 (sequence 5 and sequence 6), primer 4 (SEQ ID NO: 7 and SEQ ID NO: 8), primer pair 5 (SEQ ID NO: 9 and SEQ ID NO: 10). Using the kit provided by the invention to identify the species of the stored bean elephant is not affected by the individual growth state of the specimen and the integrity of the sample, and has the advantages of saving time, high efficiency and accuracy. The invention can be widely used in grain storage department and quarantine department.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pair of primers for identifying or assisting in the identification of stored bean elephants and a kit thereof. Background technique [0002] Bean weevils belong to Coleoptera (Coleoptera) Bean weevil family (Bruchinae), distributed in all continents of the world except Antarctica, especially in the tropical regions of Asia, Central America and South America. There are currently about 1,400 species of bean weevils recorded in the world, belonging to 58 genera. About 84% of the species of insects in this family feed on the seeds of legumes, becoming an important class of pests of cultivated legumes. A total of 18 species of bean weeds have been recorded in my country's stored products, among which mung bean callosobruchuschinensis (Linnaeus), broad bean bean Bruchus rufimanus Boheman and pea bean Bruchus pisorum (Linnaeus) are the main stored bean pests in my country; and bean bean ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/166
Inventor 刘昌燕万正煌焦春海李莉陈宏伟刘良军伍广洪
Owner INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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