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Primer Sets and Methods for Specific Amplification of the Coding Region of vkorc1 Genes of Voles and Microtus

A gene coding, technology of the genus Voles, applied in the field of primer sets for specifically amplifying the Vkorc1 gene coding region of the genus Voles and Microtus genus, which can solve the problem of unknown Vkorc1 genes, incomplete amplification, and no anticoagulation Blood rodenticide resistance and other issues

Inactive Publication Date: 2020-06-30
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The inventors found through experiments that the primers designed based on the Vkorc1 sequence of the orange-bellied prairie vole could not completely amplify the full-length sequence of the murine Vkorc1 of the genus Voles and Microtus in my country
At present, the Vkorc1 sequence and amplified primers of the common genus Microtus and Microtus genus in my country have not been reported. For Microtus maximowiczii, Microtus fortis and Microtus The Vkorc1 gene of voles that can cause serious harm, such as Lasiopodomys brandtii and brown voles (Lasiopodomys mandarinus), of the genus Voles, is still unknown, and anticoagulant rodenticides against the above-mentioned voles have not yet been seen to produce resistance. medicinal research reports

Method used

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  • Primer Sets and Methods for Specific Amplification of the Coding Region of vkorc1 Genes of Voles and Microtus
  • Primer Sets and Methods for Specific Amplification of the Coding Region of vkorc1 Genes of Voles and Microtus
  • Primer Sets and Methods for Specific Amplification of the Coding Region of vkorc1 Genes of Voles and Microtus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Genomic DNA extraction

[0029]Collect the tail (0.5-1cm) or other tissues of Vole orientalis, cut about 20mg of the sample, and place it in a 1.5mL centrifuge tube. Add 600 μl of lysate (containing 580 μl of Tris-Hcl and 20 μg of proteinase K), heat-dissolve overnight at 56°C, remove the supernatant, add 600 μl of Tris-saturated phenol, shake up and down for 5 minutes, let stand for 10 minutes, centrifuge at 12,000 r / min for 10 minutes, and take the supernatant Add 1:1 Tris saturated phenol: chloroform (300μl: 300μl), take the supernatant, add 2 times 4 ℃ pre-cooled absolute ethanol, centrifuge at 12000r / min for 10min, rinse the DNA pellet with 1mL 75% alcohol again, 12000r / min After centrifuging for 10 min, dry the DNA pellet and add 50-100 μl of TE buffer, dissolve and store in a -20°C refrigerator.

[0030] 2. Exploration of primer design and reaction conditions

[0031] (1) The Vkorc1 gene sequence (NC_022015.1) of Microtus ochrogaster was obtained from the NC...

Embodiment 2

[0047] This example is used to illustrate the specificity of the primer set of the present invention.

[0048] Black line for Rattus norvegicus (Rattus norvegicus), Bandicota indica (Muridae), Mus musculus (Mus musculus), Hamsteridae Genomic DNA of the hamster (Cricetulus barabensis), the large hamster (Tscherskia triton) of the family Muridae, the black-lined gerbil (Apodemus agrarius) of the family Muridae, and the red-tailed gerbil (Meriones libycus) of the family Muridae The primer set designed in Example 1 was used for PCR amplification. The amplification result shows ( Figure 4 ), the above-mentioned 7 different genera of mice (from left to right, each coding region is: Rattus norvegicus, Rattus norvegicus, Mus musculus, black-lined hamster, large hamster, Apodemus spp., red-tailed gerbil) No target bands were produced after PCR amplification, and the primers designed by the present invention had a better effect on the mouse species amplification of the genus Trichope...

Embodiment 3

[0050] When designing primers, because of the need to obtain the complete CDS region of different mouse Vkorc1 genes, the design of degenerate primers was initially considered. Design the merger primers online on the CODEHOP website, and the primer sequences are as follows:

[0051] vole vk1f: 5'-GCGCCTGCTGCGCNTGYTGYAC-3';

[0052] vole vk1r: 5'-CGGTGGTGCAGCAGGCRCANGCNGT-3'.

[0053] After trying various temperatures and reaction systems, it was found that the primer could not amplify the target band. Therefore, redesign the primers. Referring to the full length of the Vkorc1 gene of the orange-bellied prairie vole, primers were designed with Primer Premier5 software. As a result, only part of the Vkorc1 sequence of four species of mice could be amplified, including the first and second exon regions. However, in other mice The third exon region, which is particularly prone to resistance variants, could not be amplified.

[0054] The replaced primer sequences are as follows...

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Abstract

The invention belongs to the field of biotechnology, particularly discloses a primer group and method for specific amplification of Cricetulus and Lasiopodomys Vkorc1 gene coding sequence region. Theprimer group includes 3 pairs of primers for respectively amplifying three exons of Vkorc1 genes, the primer sequence for amplifying the first exon is shown in SEQ ID NO.1-2, the primer sequence for amplifying the second exon is shown in SEQ ID NO.3-4, and the primer sequence for amplifying the third exon is shown in SEQ ID NO.5-6. The invention provides the amplification method based on the primer group through optimizing the amplification condition, the Cricetulus and Lasiopodomys Vkorc1 genes are amplified, and a good foundation is laid for previous related research on the Cricetulus and Lasiopodomys Vkorc1 genes and resistance to drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set and a method for specifically amplifying the Vkorc1 gene coding region of the genus Microtus and Microtus genus. Background technique [0002] Anticoagulant rodenticides are currently the main chemical rodenticides for rodent control. They can combine with the VKOR enzyme in the vitamin K cycle to prevent the production of reduced vitamin K, thereby further preventing the activation of coagulation factors, resulting in Coagulation disorders. Studies have shown that anticoagulant rodenticides have resistant mice after 8-10 years, and the generation of drug resistance has greatly reduced the use efficiency of rodenticides. Therefore, in the actual rodent prevention and control process, the resistance level of the rodent population should be detected in time, and the appropriate anticoagulant rodenticide should be selected according to the resistance level, tha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6888C12N15/11
CPCC12Q1/6806C12Q1/6888C12Q2600/124C12Q2600/156C12Q2531/113
Inventor 陈燕宋英刘晓辉王大伟李宁
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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