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Multi-RPA primer and probe for simultaneously detecting NDV, IBV, H9N2-subtype AIV and detection method of multi-RPA primer and probe

A probe and multiplex technology, applied in the field of molecular biology detection, can solve the problems of taking a long time, difficult to identify clinical diagnosis, unable to provide timely guidance for epidemic diseases, etc., to achieve low cost, high sensitivity and strong specificity. Effect

Active Publication Date: 2017-10-24
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and H9N2 subtype avian influenza virus (H9N2 subtype AIV) often cause respiratory diseases in chickens in the form of mixed infection, which is difficult to distinguish in clinical diagnosis. It takes a long time to isolate and identify these diseases, and it is impossible to provide timely guidance for clinical control of these diseases. Therefore, it is urgent to find a rapid on-site detection and diagnosis technology for NDV, IBV and AIV H9N2 subtypes

Method used

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  • Multi-RPA primer and probe for simultaneously detecting NDV, IBV, H9N2-subtype AIV and detection method of multi-RPA primer and probe
  • Multi-RPA primer and probe for simultaneously detecting NDV, IBV, H9N2-subtype AIV and detection method of multi-RPA primer and probe
  • Multi-RPA primer and probe for simultaneously detecting NDV, IBV, H9N2-subtype AIV and detection method of multi-RPA primer and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Synthesis of primers and probes for detection of Newcastle disease virus

[0056] According to the comparison results of the Newcastle disease virus hemagglutinin neuraminidase (HN) gene sequence published in GenBank, select the sequence at the 587bp-621bp position in the nucleotide sequence of the Newcastle disease virus HN gene (5'-GCAGAGATCACTCACACTCACATCAGTATTTAGCAC-3') As the upstream primer of the RPA amplification reaction; the reverse complementary sequence of the 693bp-728bp position sequence in the nucleotide sequence of the Newcastle disease virus HN gene is used as the downstream primer of the RPA amplification reaction, and the 5' end of the downstream primer is used to label the organism when synthesizing the primer Biotin:

[0057] 5'-Biotin-GTTGCACTCACACTGCAAGACTTCCGATTTTGGGTG-3'.

[0058] Using the sequence at the 634bp-681bp position in the nucleotide sequence of the Newcastle disease virus HN gene as a probe, when synthesizing the probe, m...

Embodiment 2

[0060] Example 2: Synthesis of primers and probes for detection of infectious bronchitis virus

[0061]Since there are many serotypes of chicken infectious bronchitis virus strains, the present invention selects the conserved nucleus of each serotype chicken infectious bronchitis virus strain genome on the basis of comparing the genome sequences of chicken infectious bronchitis virus strains of each serotype. Nucleotide sequence, the design of primers and probes based on this conserved nucleotide sequence. Among them, the sequence at the 57bp-92bp position in the conserved nucleotide sequence (5'-CTTAACAAAACGGACTTAAATACCTACAGCTGGTCC-3') is used as the upstream primer; the reverse complementary sequence of the 204bp-242bp position sequence in the conserved nucleotide sequence is used as the downstream primer to synthesize For the downstream primer, label the 5' end of the downstream primer with biotin: 5'-Biotin-CTAGGTAGCCCAAGCAACGGATGTATAAAGTGAGACAC-3'.

[0062] Use the nucle...

Embodiment 3

[0064] Embodiment 3: The synthesis of primers and probes for detecting H9N2 subtype avian influenza virus

[0065] According to the comparison results of the M gene nucleotide sequences of the H9N2 subtype avian influenza virus published in GenBank, select the sequence at the 223bp-262bp position in the H9N2 subtype avian influenza virus M gene nucleotide sequence (5'-CACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAG-3') As an upstream primer; the reverse complementary sequence of the 552bp-558bp position sequence in the M gene nucleotide sequence is used as a downstream primer, and the 5' end of the downstream primer is labeled with biotin (Biotin) when synthesizing the primer: 5'-Biotin-CATGCCTGATTAGTGGGTTGGTGGTAGTCGCCATCTG- 3'; Use the 375bp-433bp position sequence in the M gene nucleotide sequence as a probe. When synthesizing the probe, label Digoxigenin at the 5' end of the probe sequence, and replace the base C at position 7082 with tetrahydrofuran (THF) , A 3-carbon spacer (Spac...

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Abstract

The invention discloses a multi-RPA primer and probe for simultaneously detecting a newcastle disease virus, an infectious bronchitis virus and an H9N2-subtype avian influenza virus. The multi-RPA primer and probe comprise a primer and probe for detecting the newcastle disease virus, a primer and probe for detecting the infectious bronchitis virus and a primer and probe for detecting the H9N2-subtype avian influenza virus. The multi-RPA primer and probe are high in specificity, high in sensitivity and accurate in detection result. The invention further discloses a method for simultaneously detecting the newcastle disease virus, the infectious bronchitis virus and the H9N2-subtype avian influenza virus through multi-RPA. The method comprises the steps of primer synthesis, template RNA extraction, reverse transcription, multi-PCR amplification and amplification product analysis. The detection method is simple in operation and good in stability, and a low-cost, fast and specific field diagnosis method is provided for effective detection and identification of mixed infection of common avian respiratory diseases.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a method for simultaneously detecting Newcastle disease virus (Newcastle disease virus, NDV), infectious bronchitis virus (Infectious bronchitis virus, IBV) and H9N2 subtype avian influenza virus (Avian influenza virus). virus, AIV) multiple RPA primers and probes and detection method. Background technique [0002] With the rapid development of my country's poultry industry, new changes have taken place in the prevalence of poultry diseases. The mixed infection of multiple diseases infecting one kind of poultry at the same time is becoming more and more common, which brings great difficulties to the diagnosis and prevention of diseases. Rapid on-site diagnosis of complex diseases can buy valuable time for effective disease prevention and control, and minimize losses and risks of disease spread. [0003] Conventional polymerase chain reaction (Polymerase Chain Reaction,...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q2600/16
Inventor 赵军王川庆杨霞李永涛常洪涛陈陆王新卫刘红英姚慧霞高冬生
Owner HENAN AGRICULTURAL UNIVERSITY
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