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Specific primer of I-class MHC gene for Alligator sinensis antiviral potential detection and typing method

A typing method, anti-virus technology, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc.

Active Publication Date: 2016-02-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing Chinese alligator MHC literature, the cross-amplification of the MHC gene sites is performed using universal primers, and the results can only be used for simple evolutionary analysis

Method used

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  • Specific primer of I-class MHC gene for Alligator sinensis antiviral potential detection and typing method
  • Specific primer of I-class MHC gene for Alligator sinensis antiviral potential detection and typing method
  • Specific primer of I-class MHC gene for Alligator sinensis antiviral potential detection and typing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Two pairs of primers, I1327E2F / I1327E2R and I20E3F / I20E3R, were used to amplify the MHC genes of different alligator individuals and perform SSCP typing on the PCR products:

[0056] 1. DNA extraction: Take the alligator blood sample added with EDTA anticoagulant from the -20°C refrigerator, thaw it, put 20 μL in a 1.5 mL centrifuge tube, digest it overnight, and extract the genome from the blood sample by phenol-chloroform extraction DNA. Use a spectrophotometer to detect the concentration of the extracted DNA solution, use 1% agarose gel electrophoresis with DL2000DNAmarker to detect the length range and quality of the extracted DNA fragments, take some DNA stock solution and dilute it to a dilution solution with a concentration of about 200ng / μL,- Store at 20°C.

[0057] 2. PCR amplification: using the qualified DNA dilution as a template, two pairs of primers, I1327E2F / I1327E2R and I20E3F / I20E3R designed in the present invention, are used for PCR amplification. Th...

Embodiment 2

[0069] Using I1327E3F / I1327E3R, 120E2F / I20E3R two pairs of primers to amplify the MHC genes of different alligator individuals and perform SSCP typing on the PCR products:

[0070] 1. DNA extraction: Take the alligator blood sample added with EDTA anticoagulant from the -20°C refrigerator, thaw it, put 20 μL in a 1.5 mL centrifuge tube, digest it overnight, and extract the genome from the blood sample by phenol-chloroform extraction DNA. Use a spectrophotometer to detect the concentration of the extracted DNA solution, use 1% agarose gel electrophoresis with DL2000DNAmarker to detect the length range and quality of the extracted DNA fragments, take some DNA stock solution and dilute it to a dilution solution with a concentration of about 200ng / μL,- Store at 20°C.

[0071] 2. PCR amplification: using the qualified DNA dilution as a template, two pairs of primers, I1327E3F / I1327E3R and 120E2F / I20E3R designed by the present invention, are used for PCR amplification. The PCR amp...

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Abstract

The invention relates to a single-locus specific primer of I-class main tissue compatibility compound gene sequence for amplified Alligator sinensis antiviral potention detection and a typing method. A specific sequence of the primer is shown as SEQ ID NO.1-SEQ ID NO.8. The amplified primer has stable single-locus specific amplifying capability in Alligator sinensis populations, a product obtained by amplifying the amplified primer can be used for genotyping of different individuals through subsequent single strand conformation polymorphism (SSCP) experiments, evaluation of I-class MHC gene polymorphism of Alligator sinensis individuals in the populations can be realized, potential, of the Alligator sinensis individuals, in resisting viral diseases can be detected, and important reference can be provided for new population founder selection, wildness reintroduction individual selection and artificial matching in Alligator sinensis protection work.

Description

technical field [0001] The invention discloses a plurality of pairs of single-site specific primers and a typing method for amplifying a class I major histocompatibility complex (MHC) gene sequence for detecting the anti-virus potential of Chinese alligator. Background technique [0002] Chinese alligator (Alligators inensis, Fauvel) belongs to the class Reptilia, Crocodiles, Fauvelidae, Alligator subfamily, Fauvel genus, is an ancient animal, originated in the Triassic period 230 million years ago, known as reptiles The "living fossil" in animals is a rare and endangered animal unique to my country and a national first-level key protected animal (1972). It was listed in Appendix I of the Convention on International Trade in Endangered Animals and Plants (CITES), and was also recognized by the International Union for Conservation of Nature ( IUCN) is classified as a critically endangered species and is one of the most threatened and endangered species of crocodilians in existe...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 方盛国万秋红倪小伟
Owner ZHEJIANG UNIV
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