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A method for identifying large-bone chicken breeds using enzyme digestion technology and a special primer pair for pcr amplification

An enzyme cutting technology and primer pair technology are applied in the field of specific identification of local chicken breeds in China, which can solve the problems of small number of authentic breeds, high cost, long feeding cycle, etc., and achieve the effect of convenient identification method, low cost and fast speed.

Inactive Publication Date: 2020-04-10
JIANGSU INST OF POULTRY SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The big-bone chickens have a long breeding cycle and can only be reared free-range. The cost is relatively high and the risk is relatively high. Generally, enterprises and farmers are unwilling to get involved. As a result, although Zhuanghe big-bone chickens are delicious, the number of authentic varieties is small, and fake breeds of chickens are rampant. market

Method used

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  • A method for identifying large-bone chicken breeds using enzyme digestion technology and a special primer pair for pcr amplification
  • A method for identifying large-bone chicken breeds using enzyme digestion technology and a special primer pair for pcr amplification
  • A method for identifying large-bone chicken breeds using enzyme digestion technology and a special primer pair for pcr amplification

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Experimental program
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Embodiment Construction

[0017] DNA extraction - phenol imitation method

[0018] (1) Take 500 μl of venous blood from large-bone chicken, cut it into pieces, add 500 μl of cell lysate, add 500 μl of cell lysate (0.5% sodium dodecyl sulfate (SDS), 0.1mol / L sodium chloride (NaCl), 10 mmol / L L triisopropylethanesulfonyl hydrochloride buffer (Tris-HCl, pH 8.0), 1 mmol / L ethylenediaminetetraacetic acid (EDTA, pH 8.0)) for 30 min, 10 μl of 10% proteinase K, and overnight in a water bath at 55°C.

[0019] (2) Add an equal volume of Tris-saturated phenol (pH 8.0) to the digested blood sample, slowly invert the centrifuge tube for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and carefully draw the supernatant into a clean centrifuge tube with a large-caliber pipette tip.

[0020] (3) Repeat the previous step once. If the supernatant is clear and transparent, this step can be omitted.

[0021] (4) Add an equal amount of phenol:chloroform (1:1), slowly invert the centrifuge tube for 10 minutes, centri...

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Abstract

The invention discloses a method for identifying the variety of large bone chicken by using an enzyme digestion technique and a special primer pair for PCR amplification. Special primers are shown in the sequence list SEQ ID NO 1-2. The method for identifying the variety of the large bone chicken comprises the following steps that PCR amplification and enzyme digestion are simultaneously conducted on large bone chicken sample DNA and a to-be-tested DNA mixed sample by using the special primer pair for PCR amplification; then enzyme-digested products are tested by adopting 2.0% agarose gel electrophoresis, and two electrophoresis strips (294bp, 460bp) in the agarose gel electrophoretogram of the large bone chicken sample DNA serve as a positive result; an enzyme-digested product agarose gel electrophoretogram of the to-be-tested DNA mixed sample is compared with the positive result, and if two electrophoresis strips (294bp, 460bp) appear in the enzyme-digested product agarose gel electrophoretogram of the to-be-tested DNA mixed sample, the to-be-tested sample is identified as a pure variety of the large bone chicken. According to the method for identifying the variety of the large bone chicken by using the enzyme digestion technique and the special primer pair for PCR amplification, PCR amplification and enzyme digestion test is conducted on the to-be-tested sample by using an SNP variable site, detected in a laboratory, in a d-loop region of the large bone chicken, and the designed primers and enzyme-digestion enzyme have good specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the specific identification of Chinese local chicken breeds by using an enzyme cutting method. Background technique [0002] Big-bone chicken, also known as Zhuanghe chicken, is a dual-purpose local chicken breed with outstanding characteristics of large eggs. It has a large body, strong foraging ability, many and large eggs, thick and firm eggshells, and fresh and tender meat. characteristic. The Big Bone Chicken was once one of the four major agricultural brands in China along with Mongolian Horse, Qinchuan Cattle, and Landrace Pig, and ranked second among 11 chicken breeds in the National Poultry Resource Protection Breed List. The big-bone chickens have a long breeding cycle and can only be reared free-range. The cost is relatively high and the risk is relatively high. Generally, enterprises and farmers are unwilling to get involved. As a result, although Zhuanghe bi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2565/125
Inventor 高玉时樊艳凤唐修君贾晓旭葛庆联唐梦君陈大伟张小燕陆俊贤顾荣蒲俊华
Owner JIANGSU INST OF POULTRY SCI
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