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Primers, kit and detection method for detecting genders of cotuenix coturnix
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A detection method and kit technology, applied in the field of molecular biology, can solve the problems of undisclosed PCR primer sequences, time-consuming and labor-intensive, and environmental pollution.
Inactive Publication Date: 2017-02-22
HENAN UNIV OF SCI & TECH
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However, the patent does not disclose the primer sequence for PCR amplification. At the same time, the electrophoresis method is used to detect PCR products, which is time-consuming and laborious, and is not suitable for high-throughput operations. Moreover, nucleic acid dyes are carcinogens, pollute the environment, and have certain hazards to operators.
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Embodiment 1
[0030] The primers used to detect the sex of quail were designed and synthesized according to the length difference of the quail CHD1 gene on the Z chromosome and the W chromosome. The primers are as follows:
[0031] CHDS: 5'-CCATACCTCTGATCCTTCTGC-3';
[0032] CHDAS: 5'-CAAGTTACTGATTCGTCTGCG-3'.
Embodiment 2
[0034] Kit for detecting quail sex, including: 2×SYBR qPCR mix (purchased from Beijing Suo Laibao Biotechnology Co., Ltd.) 500 μL, 10 μmol / L CHDS 50 μL, 10 μmol / L CHDAS 50 μL, 100ng / μL female quail genomic DNA 20 μL , 100ng / μL male quail genomic DNA 20μL, ddH 2 O 1mL, 1 instruction manual; primers are as follows:
[0035] CHDS: 5'-CCATACCTCTGATCCTTCTGC-3';
[0036] CHDAS: 5'-CAAGTTACTGATTCGTCTGCG-3'.
[0037] Instructions:
[0038] 1) Extract the genomic DNA of the sample to be tested;
[0039] 2) Perform qPCR amplification with primers CHDS and CHDAS, and perform melting curve analysis;
[0041] 2×SYBR qPCR mix 10μL, 10μmol / L DHDS 0.7μL, 10μmol / L CHDAS 0.7μL, 100ng / μL Genomic DNA of test sample or positive control (100ng / μL female or male quail genomic DNA) 1~2μL, ddH 2 O6.6~7.6μL, 20μL in total;
[0042] The amplification procedure is as follows:
[0043] Pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s,...
Embodiment 3
[0046]Kit for detecting quail sex, including: 2×PCR mix 500μL, 10μmol / L CHDS 50μL, 10μmol / LCHDAS 50μL, 100ng / μL female genomic DNA 20μL, 100ng / μL male quail genomic DNA 20μL, ddH 2 O 1mL, DNA Marker 2000 and 1 instruction manual; the primers are as follows:
[0047] CHDS: 5'-CCATACCTCTGATCCTTCTGC-3';
[0048] CHDAS: 5'-CAAGTTACTGATTCGTCTGCG-3'.
[0049] Instructions:
[0050] 1) Extract the genomic DNA of the sample to be tested;
[0051] 2) PCR amplification using primers CHDS and CHDAS;
[0053] 2×PCR mix 10μL, 10μmol / L DHDS 0.7μL, 10μmol / L CHDAS 0.7μL, 100ng / μL Genomic DNA of test sample or positive control genomic DNA 1μL, ddH 2 O 7.6 μL, a total of 20 μL;
[0054] The amplification procedure is as follows:
[0055] Pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing at 57°C for 3.s, extension at 72°C for 20s, 40 cycles; final extension at 72°C for 5min;
[0056] 3) The amplified product has a ...
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Abstract
The invention discloses primers, a kit and a detection method for detecting the genders of cotuenix coturnix, and belongs to the technical field of molecular biology. According to the invention, specific primers are designed by using length difference of CHD1 (Chromodomain-Helicase-DNA-binding protein 1) genes of the cotuenix coturnix on Z chromosome and W chromosome; genome DNAs of the cotuenix coturnix are taken as templates for performing PCR (PolymeraseChain Reaction) amplification and dissolutioncurve analysis; the karyotype of female cotuenix coturnix is ZW type, a PCR product contains two fragments with different sizes, and the sizes of strips are 470bp and 638bp respectively, so that the dissolution curve has two peaks; the karyotype of male cotuenix coturnix is ZZ type, a PCR product only contains one fragment, and the size of the strip is 638bp, so that the dissolution curve has one peak; the male cotuenix coturnix and the female cotuenix coturnix can be clearly distinguished by using the difference of the dissolution curve. The detection method for the genders of the cotuenix coturnix, disclosed by the invention, has the characteristics of quickness, accuracy, economy and practicality, and is suitable for high-throughput operation.
Description
technical field [0001] The invention relates to a primer for detecting quail sex, and also relates to a kit containing the primer and a method for detecting quail sex, belonging to the technical field of molecular biology. Background technique [0002] Quail (Cotuenix coturnix), also known as Japanese quail, is a high-yield and high-efficiency special bird. Quail sex is closely related to its productivity. In meat quail, male quail grows faster, and the economic benefit is much higher than that of female quail; while in egg quail, the opposite is true. At present, although the self-sex matching system has been developed by livestock and poultry workers for sex identification of quails. According to the principle of sex-linked inheritance, the male and female of the offspring can be identified according to the color of the fluff at the time of hatching, but the sex of the parents remains the same. It needs to be identified by the traditional anus turning method. However, i...
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