High-throughput kit for simultaneously detecting three avian virus diseases, and detecting method and application thereof

A kit, the technology of influenza virus, applied in the field of molecular biology detection

Active Publication Date: 2018-05-29
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] What this research will be carried out is to simultaneously identify the subtypes of IBV, NDV and AIV and their H5, H7 and H9 viruses. Based on iPDMS (initiator integrated poly(dimethysiloxane), iPDMS) as the gene chip membrane, the nucleic acid detection of the above viruses will be carried out. diagnostic methods have not yet been reported

Method used

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  • High-throughput kit for simultaneously detecting three avian virus diseases, and detecting method and application thereof
  • High-throughput kit for simultaneously detecting three avian virus diseases, and detecting method and application thereof
  • High-throughput kit for simultaneously detecting three avian virus diseases, and detecting method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] [Example 1] Design and screening of primers and probes

[0068] According to the M gene of AIV in GenBank, the hemagglutinin gene sequence of H5, H7 and H9 subtype avian influenza virus, the nucleocapsid protein gene (N gene) of IBV and the fusion protein gene (F gene) of NDV, carry out with DNAMAN software For homology analysis, use Primer Express5.0 software to design specific primers and gene probes in its conserved regions. For each probe, design 2 or more primers and probes for screening. For primers and probes with good cross-reaction and amplification efficiency, the 5' upstream of the primers are all modified with Biotin. The primers and probes of each subtype are shown in Table 2 below.

Embodiment 2

[0069] [Example 2] Preparation of positive standard

[0070] A. Extract the total RNA of IBV and NDV virus samples respectively;

[0071] B, the IBV and NDV obtained in the step A are reverse-transcribed respectively to obtain the cDNA of IBV and NDV virus;

[0072] C. Use the cDNA obtained in step B as a template, and use the nucleotide sequences shown in Table 1 below as upstream primers and downstream primers to amplify IBV and NDV virus-specific target fragments to obtain IBV-N and NDV-F Specific target fragments;

[0073] D. Connect the IBV and NDV-specific target fragments obtained in step C to the pMD18T vector and transfer them into Escherichia coli. The plasmid laboratory that connects the fragments of H5, H7, H9 and M to the pMD18T vector already exists, and the preparation process As above, the primer sequences are shown in Table 1 below, and equal volumes of the appealing plasmids were mixed to obtain positive standards with a final concentration of 0.1 mg / ml of ...

Embodiment 3

[0074] [Example 3] The kit that can simultaneously detect NDV, IBV and AIV and H5, H7, H9 subtype influenza virus thereof

[0075] Contains the following components:

[0076] (1) 48-hole gene reaction plate prepared with probes, five white films;

[0077] (2) Lotion 1 (20×SSC), volume 100mL;

[0078] (3) The volume of lotion 2 (10% SDS, 10g of SDS dissolved in water to 100ml) is 30mL;

[0079] (4) The volume of lotion 3 (1M sodium citrate) is 100mL;

[0080] (5) 5 μl of forward primer (4nmol / μl), 5 μl of reverse primer (4nmol / μl);

[0081] (6) POD (Streptavidin-Horseradish Peroxidase): 50μL;

[0082] (7) One-step multiplex RT-PCR reagents, including 2 × One Step Q Probe Mix3mL, OneStep Q Probe Enzyme Mix300μl and RNase-free water3mL in the reagent; the above-mentioned reagents are commercially available one-step multiplex RT-PCR kits (HiScript Reagents in II One Step qRT-PCR Probe Kit product number: Q222-01).

[0083] (8) Positive control sample: 200 μL of the positive ...

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Abstract

The invention discloses a high-throughput kit for simultaneously detecting three avian virus diseases, and a detecting method and the application thereof. A detecting kit for detecting NDV ((NewcastleDisease Virus), IBV (Infectious bronchitis Virus), AIV (Avian Influenza Virus) and H5, H7 and H9 subtype influenza virus nucleic acid thereof through a gene chip method provided by the invention hasa higher detecting speed, higher sensitivity and convenience in operation. The invention researches and develops a detecting kit for simultaneously detecting the NDV, the IBV, the AIV and the H5, theH7 and the H9 subtype influenza virus nucleic acid thereof, and can quickly diagnose whether chickens are infected with the detected viruses and monitor the morbidity situation of the chickens in practical production. A new and effective detecting means is provided for large-scale virus monitoring, epidemiological investigation, and prevention and control of the NDV, IBV, AIV and H5, H7 and H9 subtype influenza virus nucleic acid clinically.

Description

Technical field: [0001] The invention belongs to the field of molecular biology detection methods, and in particular relates to a kit for simultaneous detection of NDV, IBV, AIV and H5, H7, H9 subtype influenza viruses, a detection method and an application thereof. Background technique: [0002] Avian influenza (AI) is an infectious disease of birds or humans caused by influenza A virus (AIV) of the family Orthomyxoviridae. According to the difference of hemagglutinin (HA) and neuraminidase (NA) antigens of AIV, AIV can be divided into 16 HA subtypes (H1~H16) and 9 NA subtypes (N1~N9). In poultry, The main epidemics are H9N2, H5N1 and H7N3 subtype influenza viruses. Newcastle disease (ND), also known as Asian chicken plague, is a highly contagious and fatal disease that mainly affects chickens, turkeys, wild birds and ornamental birds caused by avian parainfluenza virus type Newcastle disease virus (NDV). Diseases, there are two types of strong strains and weak strains, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/6837C12R1/93
CPCC12Q1/6837C12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/501
Inventor 周继勇闫丽萍肖倩雷静
Owner NANJING AGRICULTURAL UNIVERSITY
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