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Method for rapidly detecting MD resistance chickens

A Marek's disease and resistance technology, applied in the field of Marek's disease, can solve the problems of difficult judgment, difficulty, weak electrophoresis results, etc.

Inactive Publication Date: 2013-12-25
GUANGXI UNIV
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Problems solved by technology

Nevertheless, in the detection, this technique is more difficult, because sometimes the amount of the target fragment is too small, the electrophoresis result is too weak, and it is not easy to judge

Method used

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  • Method for rapidly detecting MD resistance chickens
  • Method for rapidly detecting MD resistance chickens
  • Method for rapidly detecting MD resistance chickens

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Embodiment Construction

[0024] 1. Test steps

[0025] 1) Extraction of sample RNA

[0026] Select 4 MD resistance (BF gene) homozygous Xiayan chickens (A 11 ,C 23 ,D 8 and D 12 ) and 2 MD susceptible (BF gene) homozygous Xiayan chickens (A 5 and B 21 ) liver, spleen, heart, and PBLs (peripheral blood lymphocytes), use the Trizol RNA extraction kit to extract the total RNA of the above tissues and organs according to the product instructions, and dissolve them in 35 μL RNase-free double-distilled water, and react immediately Transcribe and synthesize cDNA.

[0027] 2) Synthesis of cDNA

[0028] Take 14.5 μL of RNA sample, add 1 μL of 25pm BF outer (inner) downstream primer, 70°C for 5min, place at -20°C for 5min; then add 5μL RT-Buffer (5×), 2μL dNTP (10mM), 200U M-MLV inversion Record enzyme, 25U Rnasin and mix well, place at 37°C for 1h, and react at 96°C for 5min, finally get 25μL cDNA, store at -20°C for later use.

[0029] 3) The first round of semi-nested PCR amplifies the alternatively...

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Abstract

The invention discloses a method for rapidly detecting MD resistance chickens. A semi-nested PCR is adopted to conduct two-round amplification on a BF gene variable splicing exon 7, and primer sequences of the two-round amplification are SEQ. ID. No. 1 and 2, and SEQ. ID. No.3 and 4 respectively. In the first-round amplification, MD easily-infected xiayan chickens A5 and B21 can be amplified to two fragments of 195 bp and 162 bp, while MD resistance xiayan chickens A11, C23, D12 and D8 can only be amplified to the segment of 195bp. In the second-round amplification, the MD easily-infected xiayan chickens A5 and B21 can be amplified to the segment of 141bp, while the MD resistance xiayan chickens A11, C23, D12 and D8 can not be amplified to the segment of 141bp. Therefore, the method can easily, rapidly and conveniently authenticate MD resistance / easily-infected haploids, and further the MD resistance / easy infection of the chickens can be authenticated.

Description

technical field [0001] The invention relates to Marek's disease, in particular to a rapid detection method for Marek's disease (Marek's disease, MD)-resistant chickens. Background technique [0002] Dalgaard et al. used PCR method to amplify white legion chicken B 21 、B 130 , BW1, B 12 、B 19 and B 15 Alternative splicing of exon 7 in haploid BF mRNA, it was found that B 12 、B 19 and B 15 These MD-susceptible haploid BF mRNAs partially lack exon 7 (i.e. BF2 mRNA lacks exon 7 but BF1 mRNA does not lack exon 7 in haploid BF), whereas B 21 and with B 21 MD resistance similar to B 130 Neither BF mRNA of , BW1 lacks exon 7 (that is, neither BF2 nor BF1 mRNA lacks exon 7 in haploid BF). Nevertheless, in the detection, this technique is more difficult, because sometimes the amount of the target fragment is too small, and the electrophoresis result is too weak, so it is not easy to judge. Contents of the invention [0003] The technical problem to be solved by the presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 韦平金元昌韦天超磨美兰黄亮崔帅
Owner GUANGXI UNIV
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