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Specific primers and typing method of class I mhc gene for detection of anti-virus potential of Chinese alligator

A typing method and anti-viral technology, applied in DNA/RNA fragments, recombinant DNA technology, etc.

Active Publication Date: 2017-06-30
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 primers and typing method of class I mhc gene for detection of anti-virus potential of Chinese alligator
  • Specific primers and typing method of class I mhc gene for detection of anti-virus potential of Chinese alligator
  • Specific primers and typing method of class I mhc gene for detection of anti-virus potential of Chinese alligator

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 DL2000DNA marker 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. T...

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 DL2000DNA marker 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 am...

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PUM

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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 multiple pairs of single-site specific primers and a typing method for amplifying a class I major histocompatibility complex (MHC) gene sequence for detecting the antiviral potential of Chinese alligator. Background technique [0002] Alligator sinensis (Alligator sinensis, Fauvel) belongs to Reptilia, Crocodiles, Sinensis, Alligator subfamily, Fauvel, is an ancient animal, originated in the Triassic period 230 million years ago, known as The "living fossil" among reptiles is a rare and endangered animal endemic to my country and a first-class national key protected animal (1972). It is listed in Appendix I of the "Convention on International Trade in Endangered Animals and Plants" (CITES), and is also recognized by the International Union for Conservation of Nature. (IUCN) classified as critically endangered species, is one of the most threatened and endangered species among extant crocodilians. At present, there are only about ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 方盛国万秋红倪小伟
Owner ZHEJIANG UNIV
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