Primers and kit for screening cows with mastitis resistance and applications thereof
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A technology of mastitis and kits, applied in the field of animal genetics and breeding, to achieve the effects of improving accuracy, reducing economic losses, and reducing morbidity
Inactive Publication Date: 2014-04-30
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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[0005] Aiming at the above-mentioned prior art, in order to solve the deficiency of the existing verification functional SNP technology, the present invention provides a method fo
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Embodiment 1
[0025] Example 1 The method for identifying the abnormal splice body of the gene caused by the functional SNP of CD46, a dairy cow mastitis resistance gene
[0026] 1.1 Identification of CD46 gene alternative splicing body
[0027] According to the GenBank accession number NM_001242561.1 sequence, use Primer Premier5 primer software to design specific primers CD46F and CD46R (as shown in SEQ ID NO.1 and SEQ ID NO.2) to amplify the CD46 gene mRNA sequence, and the PCR amplification conditions are : Denaturation at 94°C for 4 minutes; denaturation at 94°C for 30s, annealing at 52°C to 65°C for 30s, extension at 72°C for 1.4min, and this step for 35 cycles; extension at 72°C for 5min. The PCR product was detected by 2% agarose gel electrophoresis, and it was found that with the increase of the 12 return temperatures between 52°C and 65°C, in addition to the main transcript electrophoresis band, there was a larger fragment band above it, Such as figure 1 indicated by the arrow. ...
Embodiment 2
[0037] Embodiment 2 detection kit
[0038] A kit for screening anti-mastitis dairy cows based on the SNP at position 32457 of the CD46 gene, including the following reagents:
[0039] (1) A pair of special primers 3F and 3R for identifying the SNP of CD46 gene (as shown in SEQ ID NO.7 and SEQ ID NO.8);
[0040] (2) 2×Taq PCR MasterMix;
[0041] (3) FastDigest buffer;
[0042] (4) FastDigest restriction enzyme EcoRI;
[0043] (5) ddH 2 O;
[0044] (6) DNA Marker.
[0045] The concentration of 3F and 3R primers is 100 μmol / L; 2×Taq PCR MasterMix (including detection dye) is prepared by MgCl 2 (4mM), dNTPs (0.4mM), Taq DNA Polymerase (0.05units / μl).
[0046] The kit instructions include the following:
[0047] (1) Collect and extract bovine blood DNA, dilute primers to 10 μmol / L, and perform PCR reaction. Use 25 μl PCR amplification reaction system: template DNA (100ng / μl) 1 μl, upstream and downstream primers 3F and 3R each 0.5 μl, 2×Taq PCR MasterMix 12.5 μl, use ddH ...
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Abstract
The invention discloses a pair of primers for screening cows with mastitis resistance. The sequence of an upstream primer is shown in SEQ ID NO.7 and the sequence of a downstream primer is shown in SEQ ID NO.8. The invention also discloses a kit for screening cows with mastitis resistance. The kit comprises the primers and conventional reagents required for PCR (polymerase chain reaction) amplification. The primers and the kit can be used for screening cows with mastitis resistance. An application method comprises the steps of extracting genome DNA (deoxyribonucleic acid) of cows to be detected, carrying out specific PCR amplification by utilizing the primers, and detecting the PCR amplification products to judge the genotype, wherein the hybrid type has relatively strong mastitis resistance, therefore, the cow individuals with hybrid genotypes are selected for genetic molecular breeding of cows. The method has the effects of improving the accuracy of selection of cow individuals with mastitis resistance and saving the genetic improvement time of mastitis resistance of cows, and has good application prospects.
Description
technical field [0001] The invention relates to a primer, a kit for screening mastitis-resistant cows, and an application in cow mastitis-resistant breeding, belonging to the technical field of animal genetics and breeding. Background technique [0002] Dairy cow mastitis is one of the most common and complex diseases in dairy farming, causing huge losses to the dairy industry (Seegers et al., 2003). The selection of mastitis resistance candidate genes as a long-term control and prevention method can reduce the use of antibiotics in dairy cow production, improve the quality and safety of milk, and reduce economic losses. [0003] CD46 protein (membrane accessory protein) belongs to the family of complement regulatory proteins, anchored to transmembrane glycoproteins on almost all nucleated cells, it can regulate the activity of complement factors, and prevent normal cells from being damaged by complement regulation (Barilla-LaBarca et al., 2002; Hawkins et al., 2010). Acco...
Claims
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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 黄金明王秀革鞠志花齐超张燕李秋玲王长法高运东仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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