Primer composition, kit and method for identifying early sex of pigeon

A primer composition and a technology for identifying pigeons, which are applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of large animal damage, cumbersome operations, and low accuracy, and prevent false positives The effect of false negative, simple sample collection and processing, and reliable result detection

Active Publication Date: 2020-11-24
广东科贸职业学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional pigeon sex identification method is either low in accuracy, cumbersome to operate, high in cost, and relatively harmful to animals. The existing molecular identification method is a single primer that is prone to false negatives, and the result identification requires agarose gel electrophoresis. False positives due to aerosol pollution make it difficult to be accurate, simple, economical and efficient, and it is difficult to promote large-scale production

Method used

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  • Primer composition, kit and method for identifying early sex of pigeon
  • Primer composition, kit and method for identifying early sex of pigeon
  • Primer composition, kit and method for identifying early sex of pigeon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Pigeon sex identification primer specificity verification

[0054] Select two specific fragments of pigeon W chromosome EE0.6 (the corresponding amplification primers are: EE0.6-gf: 5'-TCCTATGCCTACCACCTTCCT-3', EE0.6-gb: 5'-TTGAGAAAGAATCTGACCTGAACC-3') and It has been reported that the unnamed fragment (named novel, the corresponding amplification primers are: novel_gf: 5'-AGCAGTGTGTTTACCGTATATTTCC-3', novel_gb: 5'-CGGCAGTTGCACCACTATCA-3'), extracted with the genomic DNA extraction kit Pigeon feather marrow genome is template, template amount is about 50ng, carries out conventional PCR, agarose nucleic acid gel result ( figure 1 ) shows that the amplified fragment size of the single primer novel hen (NF sample) is about 450bp, and the cock (NM) has no band; the size of the single primer EE0.6 hen (EF) is about 360bp, and the same cock (EM) has no Bands: mixed double primers noval and EE0.6 Hen pigeon (NEF) has two fragments with the same size as the respecti...

Embodiment 2

[0055] Embodiment 2: Sensitivity test result

[0056] The extracted male and female pigeon genomes were diluted and used as templates for double-primer PCR. The results showed ( figure 2 ), 0.36ng template amount can be detected in the 20ul reaction system, the sensitivity is good, the hen pigeon has a band, the male pigeon has no band, and the specificity is good.

Embodiment 3

[0057] Embodiment 3: Pigeon genome preparation effect

[0058] Use I (the method of the present invention, alkaline lysis method), II (protease cleavage method) and III (genome kit) three methods to directly crack the newborn feather feather pith to obtain the young pigeon genome, and get 5 μl of sample loading to run nucleic acid electrophoresis ( image 3 Left), add 1μl as template, 20μl system PCR amplification ( image 3 right). The method II (protease cleavage method) is to use proteinase K (RT403, Tiangen Biochemical Technology (Beijing) Co., Ltd.) according to the instructions, digest at 37°C for 3 hours, and heat at 99°C for 15 minutes to eliminate the activity of protease. Method III (genome kit) is to use the blood / cell / tissue genomic DNA extraction kit (DP304, Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genome according to the instructions.

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Abstract

The invention discloses a primer composition, a kit and a method for identifying early sex of a pigeon. The primer composition for identifying the early sex of the pigeon comprises EE0.6-gf: 5'-TCCTATGCCTACCACCTTCCT-3', EE0.6-gb: 5'-TTGAGAAAGAATCTGACCTGAACC-3', noval_ gf: 5'-AGCAGTGTGTTTACCGTATATTTCC-3' and noval_ gb: 5'-CGGCAGTTGCACCACTATCA-3'. The kit comprises the primer, a lysate A, a neutralization solution B, a premixed solution C and a nucleic acid dye. When the kit is used for detection, the detection result is reliable, the method is simple and convenient, agarose gel electrophoresisis not needed, and the nucleic acid dye directly detects the PCR result.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer composition, a kit and a method for identifying early sex of pigeons. Background technique [0002] As a late bird, pigeons cannot pass anal sex identification like ordinary poultry (chicken, duck and goose, etc.). Only when they are paired can males and females be identified, but sometimes non-sexual mating also occurs. Now there are traditional pigeon sex identification methods such as appearance and behavior characteristics, laparoscopy, fecal steroid detection and karyotype analysis. Molecular identification mainly focuses on sex chromosome-specific fragments CHD and EE0.6. The male and female pigeons were identified through the process of multiplication and agarose gel electrophoresis, and the results showed that there was 1 band of CHD gene fragment in the male pigeon and 2 bands in the hen pigeon, and there was no band in the EE0.6 fragment in the male pig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6879C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6879C12Q1/6888C12Q1/686C12Q2563/173
Inventor 刘国乾刘思伽龙航宇陈伟波孙鸿
Owner 广东科贸职业学院
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