PCR technology-based quail sex identification method
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A technology for quail and sex, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of cumbersome operation, increased economic cost, and gel electrophoresis distinction, so as to enrich existing technologies, reduce breeding costs, The effect of reducing the amount of breeding
Active Publication Date: 2016-12-21
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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The advantage of this method is that the cost is low, and it is widely used under the existing identification conditions. The disadvantage is that the operation is cumbersome, the time is long, and it is easy to cause pollution.
In recent years, some scholars have redesigned primers to distinguish ZW bands by using the different melting temperatures of CHD-W and CHD-Z amplification products, but the amplification reactions of CHD-Z and CHD-W were carried out in two PCR reaction tubes respectively. This not only increases the economic cost, but also has low practicability, and on the other hand, misjudgment may occur; another report shows that CHD-W and CHD-Z are amplified with the same primers in the same tube, and the amplified fragments are used to Z and W have 3 SNPs, and the melting temperature of their amplified products is different to distinguish. This method only uses a pair of primers, and the sequence and size of the generated fragments are very close, so they cannot be distinguished by gel electrophoresis; the disadvantages are: Users who do not have a melting curve analyzer cannot use it, and lack a simple and effective method to verify the results
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Embodiment 1
[0022] Utilize the microsatellite fragment that only exists on the quail sex chromosome W chromosome to design a pair of microsatellite primer pair, the sequence of described primer is:
[0025] In order to avoid the misjudgment of the results caused by individuals who have not amplified bands due to template problems, the present invention designed a pair of internal reference primers as a control during PCR amplification, and its nucleotide sequence is as follows (259bp):
[0026] β-actin-F1:5'-CCCTGTATGCCTCTGGTC-3'
[0027] β-actin-R1:5'-ATCTCCTGCTCGAAATCC-3
Embodiment 2
[0029] (1) Genomic DNA extraction from quail blood by kit method
[0030] Take 10 μL of quail anticoagulated blood, add 20 μL of proteinase K and 500 μL of BB3, vortex for 15 s to mix the sample thoroughly, incubate at room temperature for 10 min; briefly centrifuge, transfer the entire solution into a spin column, centrifuge at 12,000×g for 1 min, discard Filtrate; add 500 μL solution CB3, centrifuge at 12000×g for 30 s, discard the filtrate; add 500 μL solution WB3, centrifuge at 12000×g for 30 s, discard the filtrate, repeat this step once; centrifuge at 12000×g for 2 min, completely remove residual WB3 ;Transfer the spin column into a clean centrifuge tube, add 50-200 μL of preheated deionized water (PH>7) to the center of the column, let it stand at room temperature for 1 min, and centrifuge at 12,000×g for 1 min to collect the eluted DNA , stored at 4°C for later use.
[0031] (2) PCR amplification conditions
[0032] The total volume of the PCR reaction is 15 μL, incl...
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Abstract
The invention discloses a PCR technology-based quail sex identification method, and belongs to the technical field of biological detection identification. Two pairs of PCR primers used for PCR identification comprise a pair of micro-satellite primers and a pair of internal control gene (beta-actin) primers. The PCR identification method comprises the following steps: carrying out PCR amplification by adopting the quail sex identification PCR primers with the blood sample genome DNA of a young quail to be detected as a template, detecting the above obtained PCR amplification product by adopting agarose gel electrophoresis, judging the young quail to be detected as a male quail when the amplification product only has a 259 bp specific amplification band, and judging the young quail to be detected as a female quail when the amplification product has a 259 bp specific amplification band and a 114 bp specific amplification band. The method has the advantages of simplicity and convenience in operation, realization of sex discrimination in at the hatching time, rapidness in discrimination, accurate result, and avoiding of the disadvantages of long time, high breeding cost and high error rate of traditional methods.
Description
technical field [0001] The invention belongs to the technical field of biological detection and identification, and in particular relates to a method for quail sex identification based on PCR technology, which can be applied to early sex identification of baby quails. Background technique [0002] Quail is a special economic poultry and the smallest poultry breed, which has extremely high edible value and therapeutic effect. Quail eggs are rich in nutrition, high in protein and low in cholesterol. It can be seen that the breeding of quail for eggs has a good market prospect. Since the weight of quail eggs hatched is only about 7 grams, usually commercial egg substitutes use quails to distinguish males and females by feather color; and parental quails have the same coat color, so males and females cannot be distinguished when hatching, and generally have to wait for the quails to mature. It is not until about 30 days old that males and females can be distinguished by body sh...
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