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Method for detecting different subtype avian influenza viruses and special kit thereof

An avian influenza virus and primer pair technology, which is applied in biochemical equipment and methods, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve the problem of low resolution, etc. wide effect

Inactive Publication Date: 2010-06-09
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the resolution is not high, and when the nucleic acid fragments are similar in size (or the same), they cannot be distinguished, and these bands with similar molecular sizes may only appear as one band in electrophoresis.

Method used

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  • Method for detecting different subtype avian influenza viruses and special kit thereof
  • Method for detecting different subtype avian influenza viruses and special kit thereof
  • Method for detecting different subtype avian influenza viruses and special kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the construction of avian influenza virus positive plasmid

[0038] 1. Construction of H1 positive plasmid

[0039] Use QIAamp Viral RNA MiniKit to extract genomic RNA of avian influenza virus from H1 subtype avian influenza virus standard strain according to the method provided in the instruction manual. Then use the QIANGEN One Step kit operating instructions to carry out RT-PCR reaction (reaction conditions are shown in Table 2) with the primer pair of H1f ' and H1r ' in Table 1. The PCR product was recovered with a DNA recovery kit and connected to a T-vector. Sequencing was performed on the recovered RT-PCR product ligated carrier, and the sequencing results showed that the DNA shown in sequence 15 of the sequence listing (see sequence 15 of the sequence listing) was inserted into the T-vector.

[0040] 2. Construction of H3 positive plasmid

[0041] Use the QIAamp Viral RNA MiniKit to extract the genomic RNA of avian influenza virus from the H3 su...

Embodiment 2

[0054] Embodiment 2, application of two-color fluorescent multiplex PCR to detect different subtypes of avian influenza virus plasmids

[0055] 1. Multiplex PCR

[0056] The positive virus plasmids of seven subtypes were mixed as a template; in the mixture, the concentration of H1 positive plasmids was 1×10 6 The concentration of copy, H3 positive plasmid is 1.2×10 5 The concentration of copy, H5 positive plasmid is 8×10 6 The concentration of copy, H6 positive plasmid is 8×10 5 The concentration of copy, H9 positive plasmid is 1×10 7 The concentration of copy, N1 positive plasmid is 1.2×10 6 The concentration of copy, N2 positive plasmid is 6×10 6 Copy the subtype viral plasmid.

[0057] The 7 pairs of primers in Table 2 (H1f, H1r; H3f, H3r; H5f, H5r; H6f, H6r; H9f, H9r; N1f, N1r; N2f, N2r) were used for multiplex PCR. The multiplex PCR reaction system is shown in Table 4, and the cycle parameters are shown in Table 5.

[0058] The 7 pairs of primers in Table 2 (H1f, H1...

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Abstract

The invention discloses a method for detecting different subtype avian influenza viruses and a special kit thereof. The method comprises the following steps of:, carrying out multiple PCR using a primer pair B by a primer pair A by utilizing the cDNA of a biological sample to be tested as a template, carrying out agarose gel electrophoresis on products of the multiple PCR, and respectively carrying out gel scanning and analysis by a fluorescence imaging analyzer with the wavelength of 532 nm and 635 nm; if a fluorescence strip exists under the wavelength of 635nm, judging that the biological sample to be tested contains H3 subtype avian influenza virus; and if a fluorescence strip exists under the wavelength of 532nm, judging that the biological sample to be tested contains H6 subtype avian influenza virus. The primer pair A is a primer pair consisting of nucleotides represented by a sequence 11 and a sequence 12 in a sequence list, and the 5' end of the nucleotides represented by the sequence 12 is marked by Tamra. The primer pair B is a primer pair consisting of nucleotides represented by a sequence 3 and a sequence 4 in the sequence list, and the 5' end of the nucleotides represented by the sequence 4 is marked by Cy5. In the invention, the method has the advantages of high flux, high distinguishability, strong maneuverability, wide application range, time saving, test consumable saving, safety and the like.

Description

technical field [0001] The invention relates to a method for detecting different subtypes of avian influenza viruses and a special kit thereof. Background technique [0002] PCR (Polymerase Chain Reaction, Polymerase Chain Reaction) technology is a molecular biology technology developed in the 1980s, which is used to enrich specific DNA fragments. It uses DNA polymerase (DNA Polymerase) isolated from hot spring bacteria to simulate in vivo DNA replication through three-step reactions of denaturation, renaturation (annealing) and extension. It can rapidly amplify and replicate a very small amount of nucleic acid in a short period of time to obtain a large number of target DNA fragments. [0003] After PCR amplification is completed, identification is required to determine whether the expected amplified product has been obtained accurately and reliably. Agarose gel electrophoresis is the most commonly used method in laboratories, and it is simple and easy. Nucleic acid molec...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 黄新朱水芳韩雪清王慧煜侯立华
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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