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Primer composition for detecting different influenza viruses and application thereof

A primer composition and influenza virus technology, applied in the direction of recombinant DNA technology, microbial measurement/testing, biochemical equipment and methods, etc., can solve the problems of inability to detect viruses, laborious, inability to make rapid and timely diagnosis, etc.

Active Publication Date: 2014-11-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods are time-consuming and laborious, and cannot make a quick and timely diagnosis. Moreover, due to the very high mutation frequency of the virus, these methods often fail to detect the virus and cause missed detection.

Method used

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  • Primer composition for detecting different influenza viruses and application thereof
  • Primer composition for detecting different influenza viruses and application thereof
  • Primer composition for detecting different influenza viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, clinical sample detection

[0063] 1. Collection of clinical samples

[0064] Clinical cotton swab: nasal swab or throat swab. Store at 4°C and send to the laboratory for testing within 24 hours.

[0065] 2. Sample processing

[0066] Immerse the cotton swabs in 1ml of PBS solution (containing 10,000 units of penicillin and streptomycin), twist them fully, discard the swabs after wringing them out, centrifuge the solution at 8000rpm at 4°C for 5min, and take the supernatant as the sample solution. Store at ℃ for later use.

[0067] 3. Positive controls are nasal swabs or throat swabs of dogs clinically infected with H3N2 subtype canine influenza virus and experimentally infected with H3N8 subtype canine influenza virus, H1N1 / 2009 subtype influenza virus or seasonal H3N2 subtype human influenza virus . Store at 4°C, send to the laboratory for testing within 24 hours, and process according to the method in step 2. The negative control was sterilized do...

Embodiment 2

[0095] Embodiment 2, the specific detection of primer composition

[0096] 1. Total RNA extraction and reverse transcription

[0097]H3N2 subtype canine influenza virus (A / canine / Beijing / 364 / 2009(H3N2)), seasonal H3N2 subtype human influenza virus (A / Jiangxi / 262 / 05(H3N2)), H3N8 subtype canine influenza virus ( A / canine / California / 70645-4 / 2006 (H3N8)), H1N1 / 2009 subtype influenza virus (A / California / 04 / 2009 (H1N1)) and the mixture of these four viruses, canine distemper virus (CDV- WZ), canine parvovirus (CPV / BJ005 / 07) and negative swab samples (processing method is the same as step 2 in Example 1), extract the total RNA of each sample and synthesize cDNA by reverse transcription, the method is the same as the step in Example 1 Fives.

[0098] Two, PCR amplification detects the specificity of primer composition

[0099] Each cDNA in step 1 was used as a template, and the combination of the four pairs of primers in Table 1 was used as primers for PCR amplification, and double...

Embodiment 3

[0104] Embodiment 3, the sensitivity detection of primer composition

[0105] 1. Preparation of each dilution

[0106] H3N2 subtype canine influenza virus (A / canine / Beijing / 364 / 2009(H3N2)), seasonal H3N2 subtype human influenza virus (A / Jiangxi / 262 / 05(H3N2)), H3N8 subtype canine influenza virus (A / canine / California / 70645-4 / 2006 (H3N8)), H1N1 / 2009 subtype influenza virus (A / California / 04 / 2009 (H1N1)) and the mixture of these four kinds of viruses with double-antibody PBS solution (double Anti-PBS solution preparation method is as follows: Na 2 HPO 4 12H 2 02.9g, KH 2 PO 4 0.2g, NaCl 8g, KCl 0.2g, add distilled water to make up to 1L, add 10,000 units of penicillin and streptomycin) after autoclaving) to dilute, and make virus content of 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 , 1×10 0 , 1×10 -1 TCID 50 / 100 μl of each virus and the dilution of the virus mixture.

[0107] 2. Extract the total RNA of each virus and the dilution of the virus mixture respectively a...

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Abstract

The invention discloses a primer composition for detecting different influenza viruses and an application thereof. The primer composition disclosed by the invention comprises any one of primer pairs represented by the following (1)-(4): (1) a DNA molecule shown in SEQ ID No.1 and a DNA molecule shown in SEQ ID No.2; (2) a DNA molecule shown in SEQ ID No.3 and a DNA molecule shown in SEQ ID No.4; (3) a DNA molecule shown in SEQ ID No.5 and a DNA molecule shown in SEQ ID No.6; and (4) a DNA molecule shown in SEQ ID No.7 and a DNA molecule shown in SEQ ID No.8. The primer composition has positive meanings on timely diagnosing and timely treating pathogenetic canine, timely forecasting epidemic trends, preventing influenza large pandemic, and implementing large-scale canine influenza virus epidemiology monitoring.

Description

technical field [0001] The present invention relates to a primer composition and its application, in particular to a primer capable of simultaneously detecting H3N2 subtype canine influenza virus, H3N8 subtype canine influenza virus, H1N1 / 2009 subtype influenza virus and seasonal H3N2 subtype human influenza virus The primer composition and its application belong to the field of virus detection. Background technique [0002] Dogs are susceptible to a variety of influenza viruses. Due to the high contact between dogs and humans, influenza viruses may infect humans through infected dogs, threatening public health. Virological and serological surveys showed that canine H3N2, H1N1 / 2009 and human seasonal H3N2 influenza viruses were mainly prevalent in dog populations in my country. The H3N8 subtype canine influenza virus is mainly prevalent in European and American dog populations, and the possibility of its spread to Chinese dog populations is not ruled out. Once the canine i...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 孙怡朋刘金华王倩
Owner CHINA AGRI UNIV
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