Kit for rapidly and synchronously detecting nucleic acids of influenza virus A

An influenza virus, simultaneous detection technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as increased workload and detection cost, large sample demand, and easy missed detection. To achieve the effect of improving specificity, high sensitivity and improving accuracy

Inactive Publication Date: 2013-04-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently used RT-PCR and Real-time RT-PCR can usually only detect one subtype of influenza virus each time, and multiple tests are required to determine whether there is influenza virus and the type of virus, which is prone to missed detection
In the case of a large number of samples, the workload and detection cost will be greatly increased, and the sample needs to be large

Method used

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  • Kit for rapidly and synchronously detecting nucleic acids of influenza virus A
  • Kit for rapidly and synchronously detecting nucleic acids of influenza virus A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The composition and preparation of embodiment 1 type A influenza virus H1, H3, H5 and H9 subtype rapid simultaneous detection kit:

[0045] 1. Reagent composition: Trizol RNA lysate is a product of Invitrogen; Taq DNA polymerase (2U / μl), dNTPs (10mM), Mg 2+ (1.5mM), M-MLV enzyme (200U / μl) are Promega products; detection primers: H1, H3, H5 and H9 subtype influenza virus PCR primers and conserved M gene primers mixed in equal proportions; reverse transcription primers : 12 conserved nucleotides at the 3′ end of the influenza A virus genome; all primers were synthesized by Shanghai Sangon Bioengineering Company; H1, H3, H5, H9 subtypes and M positive quality control plasmids were constructed by our laboratory, save;

[0046] The relevant primer sequences are as follows:

[0047] H1 upstream primer sequence: GCCATTGCCGGTTTCATTG

[0048] Downstream primer sequence: GAAGCTGATTGCCCCCA

[0049] H3 upstream primer sequence: GGGAATGGTTGYTTCAARATATAC

[0050] Downstream ...

Embodiment 2

[0086] Example 2 Method for using the Rapid Synchronous Detection Kit for Type A Influenza Virus H1, H3, H5 and H9 Subtypes

[0087] 1. Sample processing

[0088] Specimens involved in the detection of influenza A virus include respiratory samples (nasal / pharyngeal swabs, sputum, nasopharyngeal aspirate, bronchoalveolar lavage fluid, etc.), serum in the acute phase, serum in the recovery phase, thoracentesis, etc.; Specimens also involve various tissues.

[0089] (1) If the sample contains a small amount of mucus (nasal / pharyngeal swab, nasopharyngeal aspirate, bronchoalveolar lavage fluid, etc.), put the sample in a centrifuge at 4°C and 2000rpm for 20 minutes to remove impurities. After centrifugation, gently open the centrifuge tube in a safety cabinet, pipette the supernatant into an EP tube for RNA extraction.

[0090] (2) If the sample liquid contains a large amount of mucus (sputum, etc.), add 1% trypsin solution (pH7.6) at a volume ratio of 1:1, digest at 25°C for 15-3...

Embodiment 3

[0106] Example 3 Application of Rapid Synchronous Detection Kit for Type A Influenza Virus H1, H3, H5 and H9 Subtypes

[0107] In the following experiments, Y is A and R is C in the degenerate primers used.

[0108] 1. Sensitivity test

[0109] 10-fold serial dilutions of influenza A virus H1N1, H3N2, H5N1, and H9N2 subtypes 0 ~1.0×10 8 copies / ml) as a template, added to the PCR reaction solution of the detection kit, carried out the amplification reaction on the PCR instrument according to the amplification reaction conditions during the sample detection, and the PCR amplification products were analyzed by 3% agarose gel electrophoresis. It has been confirmed by repeated experiments for more than 3 times that the sensitivity of the kit for detecting H1 and H9 subtype influenza viruses is 10 copies / μl; the sensitivity for detecting H3 and H5 subtype influenza viruses is both 100 copies / μl.

[0110] 2. Specificity test

[0111] Influenza A virus H1, H3, H5 and H9 subtype ra...

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Abstract

The invention provides a kit for rapidly and synchronously detecting nucleic acids of influenza virus A, which comprises five pairs of primers shown as SEQ ID No.1-10 in a sequence table and preferably comprises a primer shown as SEQ ID No.11. The kit in the invention can rapidly detect the influenza virus A existing in a sample, realizes rapid single-tube and synchronous detection and parting onsub-influenza viruses H1, H3, H5 and H9, has the advantages of good specificity, high sensitivity, short time consumption, accurate detection and the like, is suitable for monitoring epidemic situations of human and avian influenzas and rapidly identifying and diagnosing patients in the pandemic periods of the influenzas, thereby having great value in clinic application.

Description

technical field [0001] The invention belongs to the field of viral nucleic acid detection, and relates to the detection of type A influenza virus, especially the rapid and simultaneous detection and typing of H1, H3, H5 and H9 subtype nucleic acids. Specifically, the invention relates to detection primers and methods for type A influenza virus. The kit is suitable for surveillance of human and avian influenza and rapid differential diagnosis of patients during influenza pandemic. technical background [0002] Influenza is an acute and highly contagious viral disease common to humans and various animals. It is caused by Influenza Virus infection and is mainly transmitted through air droplets and contact. It is distributed worldwide and circulates repeatedly. Bring great harm to human health and livestock and poultry breeding industry. [0003] Influenza viruses belong to the Orthomyxoviridae family and can be divided into three types: A, B, and C. Among them, according to t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 裴增林吴玲潘兹书
Owner WUHAN UNIV
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