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Primer group for carrying out dual nanometer PCR (Polymerase Chain Reaction) detection on H7 and N2 subtype avian influenza virus, kit and method

A bird flu virus and primer set technology, applied in the field of molecular biology, to achieve strong specificity, high detection sensitivity, and easy operation

Pending Publication Date: 2020-08-11
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of animal viruses by nano-PCR technology has been reported, but the nucleic acid detection technology of double nano-PCR (nano-dPCR) technology reaction to simultaneously amplify H7 and N2 genes to detect and identify H7N2 subtype AIV has not been reported.

Method used

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  • Primer group for carrying out dual nanometer PCR (Polymerase Chain Reaction) detection on H7 and N2 subtype avian influenza virus, kit and method
  • Primer group for carrying out dual nanometer PCR (Polymerase Chain Reaction) detection on H7 and N2 subtype avian influenza virus, kit and method
  • Primer group for carrying out dual nanometer PCR (Polymerase Chain Reaction) detection on H7 and N2 subtype avian influenza virus, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Primer Development and Synthesis

[0027] Comparing the nucleotide sequences of the H7 subtype AIV HA gene and the N2 subtype AIV NA gene, screening the specific conserved regions of the two target genes, designing and screening 2 pairs of specific primers for the two target genes respectively, Including primer H7-F, primer H7-R, primer N2-F, primer N2-R. The sequence of primer H7-F is shown in SEQ ID NO.1, the sequence of primer H7-R is shown in SEQ ID NO.2, the sequence of primer N2-F is shown in SEQ ID NO.3, the sequence of primer N2-R is shown in The sequence is shown in SEQ ID NO.4. Primers were synthesized by Thermo Fisher Guangzhou Branch.

Embodiment 2

[0028] The establishment and optimization and specificity test of embodiment 2 double nano-PCR reaction system

[0029] 1. Strains: AIV strains H1N1, H3N6, H6N6, H9N2, NDV, IBV, ARV, ILTV and ChPV are all preserved by Guangxi Key Laboratory of Veterinary Biotechnology; H14N5, H15N9, H16N3, H13N6, H7N2 and H7N9 subtype AIV The cDNA templates of AIV of H4N5 and H5N1 subtypes were donated by Connecticut State University; the other AIV (H2N3, H8N4, H10N3, H12N5 and H11N3) strains or cDNA templates were donated by Hong Kong University.

[0030] 2. Main reagents and instruments: Nano-PCR kit (NPK02) was purchased from Shanghai Huzheng Biotechnology Co., Ltd.; DNA / RNA co-extraction kit, small plasmid extraction kit, gel recovery kit, competent cells, PCR The product quick ligation vector and DNA Marker were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the reagents M-MLV, random primers, dNTP and RNA inhibitors were purchased from Bao Biology (Beijing) Co., Ltd.; the PCR...

Embodiment 3

[0038] Embodiment 3 sensitivity test

[0039] Prepare initial concentrations of 6 x 10 8 and 6×10 8 Copy / μL of H7-T and N2-T (i.e. recombinant plasmid template), after ddH 2 O 10-fold serial dilution, take the recombinant plasmid template of each dilution, use the above-mentioned optimized double nano-PCR detection method to perform double nano-PCR and double ordinary PCR detection, and verify the sensitivity of the detection method.

[0040] The PCR product was electrophoresed through agarose gel, and the electrophoresis of the double nano-PCR result was Figure 4 , the electropherogram of the double common PCR result is Figure 5 . exist Figure 4 and Figure 5 The lanes in the middle are: M lane. DNA Marker 100bp DNA Ladder; the concentration of H7-T plasmid in lanes 1 to 9 is 6×10 8 To 6 copies / μL, the concentration of N2-T plasmid was 6×10 8 To 6 copies / μL; 10 swimming lanes. Negative control.

[0041] From Figure 4 and Figure 5 It can be seen in the results ...

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Abstract

The invention provides a primer group for carrying out dual nanometer PCR (Polymerase Chain Reaction) detection on an H7 and N2 subtype avian influenza virus, a kit and a method. The invention aims atthe specific primers of H7 and N2 subtype AIV (avian influenza virus) genes to experiment and screen different primer combinations to obtain a primer combination with a good amplification effect; then, a ratio, an annealing temperature and amplification time between primer concentration and a primer in a reaction system can be simultaneously optimized; and finally, a method capable of simultaneously detecting the H7 and N2 subtype avian influenza virus is finally established. The primer and the detection method can simultaneously determine a mixed infection and single infection situation of the H7 and N2 subtype AIV in a sample through one-time nanometer PCR reaction, especially, and a novel PCR technology, i.e., the nanometer PCR, is adopted and is more sensitive than common PCR and moreconvenient and quicker than real-time fluorescent quantitation PCR. The method simultaneously has the advantages of high specificity, high detection sensitivity, high speed, convenience and the like,is easy to operate, is very suitable to be popularized in grassroots units, and provides technical support for monitoring, preventing and controlling the H7 and N2 subtype avian influenza virus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a primer set, a kit and a method for double nano-PCR detection of H7 and N2 subtype avian influenza viruses. Background technique [0002] Avian influenza is a highly contagious disease caused by avian influenza virus (AIV), which continues to spread around the world. Avian influenza virus is a member of the family Orthomyxoviridae, and AIV can be divided into 18 different HA subtypes (H1~ H18) and 11 different NA subtypes (N1~N11). HA and NA can be combined freely, theoretically there are 198 subtypes of influenza virus. In addition, AIV can be divided into highly pathogenic AIV (HPAI) and low pathogenic AIV (LPAI) according to its pathogenicity. [0003] There are nine different subtype combinations of H7 subtype viruses, and the known subtypes include H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9. Most H7 viruses found in wild birds and poultry around th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/155C12Q2537/143
Inventor 谢芝勋李孟李丹谢丽基罗思思张艳芳张民秀黄娇玲范晴王盛谢志勤邓显文曾婷婷
Owner GUANGXI VETERINARY RES INST
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