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Kit for real-time fluorescence RT-PCR detection of H9 subtype avian influenza virus

A bird flu virus and kit technology, applied in the field of real-time fluorescent RT-PCR test kits for H9 subtype bird flu virus, can solve the problems of poor sensitivity and specificity, can not accurately reflect whether it is infected or carries the virus, and achieves effective curative effect The effect of monitoring

Active Publication Date: 2010-06-16
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, immunological methods are relatively fast and simple, but have poor sensitivity and specificity (Boivin G, Hardy I, Kress A. Evaluation of a rapid optical immunoassay for influenza viruses (FLU OIA test) in comparison with cell culture and reverse transcription-PCR .J Clin Microbiol, 2001, 39: 730-732), and the result of antibody detection only shows that the tested birds or animals have been infected with avian influenza virus, but cannot accurately reflect whether they are currently infected or carry the virus. Therefore, in terms of application has certain limitations
Especially currently poultry are mostly vaccinated and therefore cannot be screened using this method

Method used

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  • Kit for real-time fluorescence RT-PCR detection of H9 subtype avian influenza virus
  • Kit for real-time fluorescence RT-PCR detection of H9 subtype avian influenza virus
  • Kit for real-time fluorescence RT-PCR detection of H9 subtype avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Development of one-step real-time fluorescent RT-PCR detection reagent for H9 subtype avian influenza virus

[0030] 1. Design of primers and probes: by comparing and analyzing the existing AIV-H9 nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in domestic and foreign published documents, the matrix protein-encoding gene (M The conserved fragment of gene) is the amplification target site, select a highly conserved segment with no secondary structure, and use software to manually design multiple pairs of primers and probes according to the basic principles of primer and probe design.

[0031] 2. Selection of samples: According to relevant domestic and foreign literature reports, throat swabs, cloacal swabs, muscle or organ samples, serum or plasma samples can be selected.

[0032] 3. Establishment and optimization of the reaction system

[0033] Sample preparation: 3 AIV-H9 samples identified as positive by virus culture...

Embodiment 2

[0043] Example 2: One-step real-time fluorescent RT-PCR detection kit for H9 subtype avian influenza virus and its use

[0044] 1. Prepare a kit including the following components: 1 tube of RNA extraction solution (50ml / tube), 24 tubes of RT-PCR amplification reaction solution (20μl / tube), 1 tube of negative quality control (100μl / tube), 1 tube of positive Quality control substance (100μl / tube) 1 tube, quantitative reference substance (50μl / tube) 4 tubes.

[0045] 2. Specimen collection, transportation and storage

[0046] 2.1 Applicable sample types: throat swabs, cloacal swabs, muscle or organ samples, serum or plasma, etc.

[0047] 2.2 Sample collection and pretreatment (note aseptic operation)

[0048] 2.2.1 Live poultry samples—take throat swabs, cloacal swabs or fresh feces swabs. The specific collection methods are as follows:

[0049] 1) Throat swab: When taking it, the swab should be deep into the throat and upper palate and scraped back and forth 3 to 5 times to ...

Embodiment 3

[0066] Example 3: Quantitative detection of clinical samples using one-step real-time fluorescent RT-PCR detection kit for H9 subtype avian influenza virus

[0067] All experimental procedures were completed in the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, and clinical samples were provided by the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, including throat swabs, cloacal swabs, muscle or organ samples and other sample types; specimens RNA extraction, RT-PCR reaction and result analysis were carried out with reference to Example 2.

[0068] After the RT-PCR reaction is over, adjust the analysis parameters according to the amplification curve, so that the standard curve of the quantitative reference product under the standard curve (Std curve) window reaches the best (that is, the square of the correlation coefficient (R 2 )>0.97), and then analyze the clinical samples. The test results of ...

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Abstract

The invention relates to a kit for real-time fluorescence RT-PCR detection, in particular to a kit for early and rapid diagnosis of the infection of an H9 subtype avian influenza virus by using the one-step real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) technology. Since the kit has high sensitivity and specificity, the rapid and early detection and quantitative analysis of the H9 subtype avian influenza virus in samples, such as the throat swab, the cloacae swab, the muscle or the viscera sample, the sera or the plasma and the like, can be realized by the kit.

Description

technical field [0001] The present invention relates to a kind of real-time fluorescent RT-PCR detection kit of H9 subtype avian influenza virus, especially relate to the early and rapid diagnosis of H9 subtype bird by one-step real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) technology Kit for influenza virus infection. The kit has high sensitivity and specificity, and the rapid early detection of H9 subtype avian influenza virus in samples such as throat swabs, cloacal swabs, muscle or organ samples, serum or plasma can be realized by the kit of the present invention. detection and quantitative analysis. Background technique [0002] Avian influenza (Avian Influenza, AI) is the abbreviation of avian influenza, also known as true chicken plague or European chicken plague, is an acute infectious disease with multiple symptoms ranging from respiratory diseases to severe hemorrhagic lesions of systemic tissues. In 1878, Perroncito first discover...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李明邓中平高秀洁王秋泉李杰胡守旺程钢何蕴韶
Owner DAAN GENE CO LTD
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