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Ring-necked pheasant early sex determination method based on PCR technology

A technology for ring-necked pheasants and male ring-necked pheasants, which is applied in the field of early sex identification of ring-necked pheasants based on PCR technology, can solve the problems of non-existence and incompletely consistent CHD1 gene sequences, so as to enrich existing technologies and reduce breeding costs , the effect of reducing the amount of breeding

Inactive Publication Date: 2019-09-20
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the CHD1 gene sequences of different species of birds are not completely consistent, so there is no universal primer pair

Method used

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  • Ring-necked pheasant early sex determination method based on PCR technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Utilize the specific CHD1 gene segment on the ring-necked pheasant sex chromosome Z / W chromosome to design a pair of primers, the sequence of the primers is:

[0028] Upstream primer: 5'-TACTGATTCGTCTACGAGA-3';

[0029] Downstream primer: 5'-ATTGAAATGATCCAGTGCTTG-3';

[0030] In order to avoid misjudgment caused by unamplified bands due to template problems, the present invention designed a pair of internal reference primers as a control when performing PCR amplification, and its nucleotide sequence is as follows (450bp):

[0031] Upstream primer: 5'-TGGATGATGATATTGCTGC-3';

[0032] Downstream primer: 5'-ATCTTCTCCATGTCATCCC-3'.

Embodiment 2

[0034] (1) Extraction of Genomic DNA from Blood of Ring-necked Pheasant by Kit Method

[0035] Using the Tiangen Blood DNA Extraction Kit, take 10 μl of ring-necked pheasant anticoagulated blood, add 20 μl of proteinase K and mix well. Add 200 μl buffer GB, mix the sample thoroughly, incubate at 56°C for 10 min, add 200 μl absolute ethanol, and mix well by inversion. Transfer the whole solution into the adsorption column, centrifuge at 12000rpm for 30s, discard the filtrate; add 500μl buffer GD, centrifuge at 12000rpm for 30s, discard the filtrate

[0036] Add 500μl solution PW, centrifuge at 12000rpm for 30s, discard the filtrate, repeat this step once; centrifuge at 12000rpm for 2min; transfer the adsorption column into a clean centrifuge tube, add 50-200μl of elution buffer TB in the center of the column, and let stand at room temperature Centrifuge at 12,000 rpm for 1 min, collect the eluted DNA, and store at 4°C for later use.

[0037] (2) PCR amplification conditions ...

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Abstract

The invention discloses a ring-necked pheasant early sex determination method based on the PCR technology and belongs to the technical field of biological monitoring and identification. Two pairs of PCR primers for the PCR identification of the method include a pair of CHD1 gene fragment primers and a pair of reference gene primers. The method includes: using the blood sample genome DNA of a to-be-determined ring-necked pheasant as the template, using the ring-necked pheasant early sex determination PCR primers to perform PCR amplification, using agarose gel electrophoresis to detect PCR amplification products, determining that the to-be-determined ring-necked pheasant is a male ring-necked pheasant when a 590bp specific amplification band exists in the amplification product, and determining that the to-be-determined ring-necked pheasant is a female ring-necked pheasant when a 590bp specific amplification band and a 442bp specific amplification band exist in the amplification product. The method is simple and convenient in operation, capable of performing sex determination during hatching, fast in determination, accurate in result and the like and is capable of avoiding the defects that a traditional method is high in time consumption, high in feeding cost and prone to mistake making.

Description

technical field [0001] The invention belongs to the technical field of biological detection and identification, and in particular relates to a method for early sex identification of ring-necked pheasants based on PCR technology. Background technique [0002] Ring-necked pheasant (American colorful pheasant) is a meat-and-egg variety with the characteristics of plump body, high egg production, large body weight, excellent meat quality, strong disease resistance, good consistency, and stable genetics. It belongs to the special economy Poultry has extremely high edible value and ornamental and hunting effects. The breeding of ring-necked pheasants for eggs also has a good market prospect. Since the ring-necked pheasant cannot distinguish between male and female when hatching, it is necessary to wait for the ring-necked pheasant to grow to a sufficient age to distinguish between male and female by body shape and appearance, which greatly increases the breeding cost of ring-neck...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6879C12Q1/686
CPCC12Q1/686C12Q1/6879C12Q2600/166C12Q2545/101C12Q2565/125
Inventor 何川孟和肖璐杨凌宇徐珂丁金梅周浩郑煜明韩铖潇罗怀希
Owner SHANGHAI JIAO TONG UNIV
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