Specific primers and typing method of class I mhc gene for the detection of antiviral potential of crested ibis

A genotyping method and technology of a genotyping method, applied in the field of genotyping, can solve the problems of low reproductive ability, serious inbreeding, high death and disability rate of young birds, etc.

Active Publication Date: 2016-06-29
ZHEJIANG UNIV
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Problems solved by technology

After decades of protection work, although the population of crested ibises is gradually recovering, it is still not out of the endangered situation. Since the known individuals in the world are all descendants of two pairs of crested ibises rediscovered in 1981, inbreeding is serious. There are problems such as low reproductive ability and high rate of death and disability of juvenile birds

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  • Specific primers and typing method of class I mhc gene for the detection of antiviral potential of crested ibis
  • Specific primers and typing method of class I mhc gene for the detection of antiviral potential of crested ibis
  • Specific primers and typing method of class I mhc gene for the detection of antiviral potential of crested ibis

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example 1

[0044] 1. Sample preparation: Crested ibis blood and tissue samples came from artificial populations such as Deqing, Sado, Beijing, Henan, Yangxian and Louguantai.

[0045] 2. DNA extraction: phenol-chloroform method to extract blood and tissue DNA.

[0046] 3. Primer synthesis: According to the obtained crested ibis MHC gene sequence, design universal primers to amplify exon 2 for the typing of UAA loci, and BGI synthesizes primers. The primer sequences are as follows:

[0047]

[0048] 4. Primer amplification: the above-mentioned amplification primers are respectively applied to PCR to amplify the extracted sample DNA. Set the PCR thermal cycle annealing temperature range to 61°C.

[0049] 5. Amplification result detection: take 2 ul of the above-mentioned PCR amplification products and carry out agarose gel electrophoresis with a gel concentration of 1.5%. The result is as figure 1 As shown, this primer can effectively amplify the target band, and the fragment size o...

example 2

[0063] 1. Sample preparation: Crested ibis blood and tissue samples came from artificial populations such as Deqing, Sado, Beijing, Henan, Yangxian and Louguantai.

[0064] 2. DNA extraction: phenol chloroform method to extract blood and tissue DNA.

[0065] 3. Primer synthesis: Design specific primers for exon 3 of the UBA site based on the obtained Crested Ibis MHC gene sequence, and BGI synthesized the primers. The primer sequences are as follows:

[0066]

[0067] 4. Primer amplification: apply the above amplification primers to PCR respectively to amplify the extracted sample DNA. Set the PCR thermal cycle annealing temperature range to 61°C.

[0068] 5. Amplification result detection: take 2 ul of the above-mentioned PCR amplification products and carry out agarose gel electrophoresis, the gel concentration is 1.5%. The result is as figure 2 As shown, this primer can effectively amplify the target band, and the fragment size of the target band is 150 bp. The PCR ...

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Abstract

The invention relates to a set of primers for amplifying classic locus sequences of MHC class I genes of crested ibis and a genotyping method, wherein the sequences of the primers are as shown in SEQ ID NO. 1-SEQ ID NO. 10. By virtue of the amplification primers disclosed by the invention, the nucleotide sequences of various class loci of class I MHC of the crested ibis are effectively amplified, and each individual is subjected to genotyping by virtue of subsequent SSCP-HD (single-strand conformation polymorphism-HD) or sequencing technology. The specific primers disclosed by the invention have the advantages of being strong in specificity, and simple and rapid in operating process; and by typing the classic loci of the class I MHC genes of the crested ibis, the polymorphism levels of the classic loci of the class I MHC genes of the crested ibis individual are assessed and the potential of the crested ibis in resisting viral diseases is detected, so that an important reference is provided for such operations of new population founder selection, reintroduction individual selection, artificial mating selection and the like in crested ibis protection work.

Description

technical field [0001] The invention provides primers for amplifying the gene sequence of each site of MHC class I of the crested ibis and a genotyping method based on the single-strand conformation polymorphism (Single-Strand Conformation Polymorph-phism, SSCP) technology. Background technique [0002] Crested ibis, an endangered species in the world and a national and key protected animal, belongs to the genus Crested Ibis of the Ibis family of the order Aves. It is a medium-sized wading bird that was once widely distributed in China, Russia, Japan and the Korean Peninsula in East Asia. At the end of the 19th century and the beginning of the 20th century, due to human activities and other reasons, the population of crested ibises decreased significantly, and wild individuals in Russia, the Korean Peninsula and Japan became extinct one after another. Until 1981, researchers rediscovered 7 wild crested ibises in Qinling Mountains, Yang County, Shaanxi Province, my country. Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2565/125C12Q2565/131
Inventor 方盛国万秋红蓝泓
Owner ZHEJIANG UNIV
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