Reagent kit for detecting Hexi Cashmere goat abortion disease resistance allele and application method

A technology for detection kits and alleles, applied in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., can solve problems such as drug residues and difficult-to-control disease conditions, and achieve sensitive, easy-to-operate, and specific strong effect

Inactive Publication Date: 2013-08-21
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the domestic goat abortion disease mainly adopts drug prevention and control measures, which is difficult to control the disease and there is a risk of drug residues

Method used

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  • Reagent kit for detecting Hexi Cashmere goat abortion disease resistance allele and application method
  • Reagent kit for detecting Hexi Cashmere goat abortion disease resistance allele and application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: A PCR-SSCP detection kit for the resistance allele of Hexi cashmere goat abortion disease, which includes the first PCR reaction solution, the second PCR reaction solution, GOLA-DRB1*11 and GOLA-DRB1*03 DNA standard sample; SSCP detection reagent, deionized water, 10% ammonium persulfate, denaturing loading buffer, TEMED (tetramethylethylenediamine), 12% non-denaturing polyacrylamide (Acr:Bis = 37.5:1) .

[0052] The loading denaturing buffer includes 98% deionized formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 10mmol / L EDTA pH 8.0.

[0053] The reaction solution of the first PCR reaction solution: the total volume is 20 μL, of which the 10× buffer buffer is 2.0 μL, Mg 2 + The concentration is 2.5mM, the final concentration of dNTPs is 100μM, primers DRB1.1 and GIo are 0.1μM each, Taq polymerase is 0.5U, template DNA is 50ng, ddH 2 O make up the volume to 20 μL.

[0054] The reaction solution of the second PCR reaction solution: a total volum...

Embodiment 2

[0065] Embodiment 2: A method for using a Hexi cashmere goat abortion disease resistance allele PCR-SSCP detection kit, characterized in that the steps are:

[0066] (1) Mix the genomic DNA to be tested (1μL / 50ng) with the first PCR amplification solution in the kit, and amplify according to the above-mentioned first set of primer PCR reaction conditions;

[0067] (2) The second PCR amplification solution in the kit is mixed with the PCR amplification product of step (1), and amplified according to the above-mentioned second set of primer PCR reaction conditions;

[0068] (3) Use the PCR product amplified in step (2) and the DNA standard sample of GOLA-DRB1*03 or GOLA-DRB1*11 to mix with the components of the SSCP detection reagent in the kit for SSCP detection;

[0069] (4) The samples whose band pattern detected in step (3) was consistent with the standard transect pattern of GOLA-DRB1*03 or GOLA-DRB1*11 were individuals carrying the abortion resistance allele of Hexi cashme...

Embodiment 3

[0070] Example 3: A PCR-SSCP detection method for the resistance allele of Hexi cashmere goats to abortion disease, which is characterized in that the steps are: (1) Sample collection: collect Hexi cashmere goats with normal phenotype and abortion disease, and collect blood from the jugular vein 10ml, anticoagulated with ACD, frozen at - 20 ℃;

[0071] (2) Genomic DNA extraction: Genomic DNA was extracted from frozen blood samples by conventional phenol-chloroform extraction;

[0072] (3) Polymerase chain reaction:

[0073]The first round of PCR reaction system: the total volume is 20 μL, of which 10×buffer buffer is 2.0 μL, Mg 2 + The concentration was 2.5mM, the final concentration of dNTPs was 100μM, and the primers DRB1.1 and GIo Each 0.1μM, Taq polymerase 0.5U, template DNA 50ng, ddH 2 The volume was supplemented with O to 20 μL; reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 60°C for 2 min, extension at 72°C for...

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Abstract

The invention relates to the field of goat molecular marker assisted breeding in the technical field of animal biology, and particularly relates to a reagent kit for detecting a Hexi Cashmere goat abortion disease resistance allele. The reagent kit for detecting the Hexi cashmere goat abortion disease resistance allele polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) is characterized by comprising first PCR reaction liquid, second PCR reaction liquid, deoxyribonucleic acid (DNA) standard samples of GOLA-DRB1*03 and GOLA-DRB1*11, a single strand conformation polymorphism (SSCP) detection reagent, deionized water, 10% ammonium persulfate, sample degeneration buffer solution, tetramethylethylenediamine (TEMED) and 12% of non-denaturing polyacrylamide gel with Arc:Bis=37.5:1. The sample degeneration buffer solution comprises 98% of deionized formamide, 0.025% of bromophenol blue, 0.025% of xylene cyanol and 10mmol / LEDTApH8.0. The reagent kit for detecting Hexi Cashmere goat abortion disease resistance allele and the application method have the advantage that molecular marker seed selection of Hexi Cashmere goat abortion diseases of the reagent kit is characterized by being rapid, high in sensibility, low in cost and the like.

Description

technical field [0001] The invention relates to a goat molecular marker assisted breeding technology in the field of animal biotechnology, in particular to a detection kit and a detection method for abortion disease resistance alleles of Hexi cashmere goats. Background technique [0002] Abortion disease in sheep is an endemic subacute infectious disease caused by chlamydia, toxoplasma gondii, brucella infection and other factors, characterized by mass abortion of sheep. The incidence of abortion in Hexi cashmere goats is relatively high, with an average abortion rate of 25.41% and a maximum of 83.33%. [0003] Animal major histocompatibility (gene) complex (major histocompatibility complex, MHC) is closely related to a variety of diseases, and is currently the preferred locus or candidate gene for disease resistance breeding. The structure and function of MHC genes of goats are very similar to those of sheep and cattle, and have a high degree of sequence homology, and are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N27/447
Inventor 罗玉柱王继卿刘秀马小军李少斌王佳泰胡江
Owner GANSU AGRI UNIV
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