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A kind of influenza A virus detection primer, probe and kit thereof

A type of influenza A virus and kit technology, applied in the field of nucleic acid amplification, can solve the problems of many false negatives, loss of clinical significance, long time-consuming virus isolation and culture, etc.

Active Publication Date: 2020-12-18
MUDANJIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are many methods for detecting common influenza viruses, but all of them have deficiencies. For example, the electron microscope detection method is complicated and expensive, the virus isolation and culture takes a long time and there are many false negatives, and the clinical significance is usually lost after the culture results are obtained. The method detects virus-specific antibodies but can only detect one virus at a time
However, the above method is difficult to achieve uniform detection of different subtypes of influenza virus

Method used

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  • A kind of influenza A virus detection primer, probe and kit thereof
  • A kind of influenza A virus detection primer, probe and kit thereof
  • A kind of influenza A virus detection primer, probe and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the method for using the kit for detecting influenza virus

[0021] (1) Nucleic acid extraction: take clinical samples to be tested, and extract viral nucleic acid;

[0022] (2) Constant temperature amplification: Mix 15 μl constant temperature amplification buffer, 10 μl constant temperature amplification enzyme solution, and 25 μl clinical sample solution obtained in step (1) to form a 50 μl reaction solution, vortex and oscillate evenly, and inject the primer set and probe In the amplification reactor; place the reactor in a constant temperature amplification instrument, and react at 40°C for 50min;

[0023] (3) Result determination: After the reaction is over, determine whether the sample liquid to be tested contains the genome of the corresponding influenza virus according to the amplification curve of the sample in each reaction chamber; if the sample to be tested produces an S-type amplification curve, Then the sample to be tested contains the geno...

Embodiment 2

[0024] Embodiment 2, the detection of actual clinical sample

[0025] 1. Extraction of viral nucleic acid from clinical samples

[0026] Viral nucleic acid extraction from clinical samples was carried out using commercial kits, such as QIAamp Viral RNA Mini Kit (Qiagen), Trizol (Invitrogen), and viral RNA extraction was carried out according to the instructions;

[0027] 2. Amplification reaction system preparation

[0028]Take 15 μl of constant temperature amplification buffer, 10 μl of constant temperature amplification enzyme solution, and 25 μl of the RNA solution extracted in the above step 1, mix them into 50 μl of reaction solution, vortex and oscillate evenly, and inject into the constant temperature amplification reaction chip loaded with primers and probes;

[0029] The solvent of the constant temperature amplification buffer is sterile water, and the solute and concentration are as follows: 250mM Tris-HCL with pH 8.0, 80mM DTT, 15mM dNTP, 15mM rNTP, 60mM MgCl 2 , ...

Embodiment 3

[0033] Embodiment 3 comparative example setting

[0034] In the experiment, purchased commercial kits A and B (wherein, the kit is a RT-PCR detection kit, and kit B is a colloidal gold method detection kit) were used to verify the reliability of the detection results of the kit of the present invention. For the test kit of the present application is positive, while other comparative examples (kit A or B) are negative results, subsequent re-sampling of these sample cases is carried out for various test verifications. At the same time, the method of the present application is also compared to the clinically detected influenza B virus samples.

[0035] The clinical samples used in this application are all from hospitals in Heilongjiang Province, with a total of 783 cases. The test results are shown in Table 1 below:

[0036] Table 1: Comparison between the kits of this application and commercial kits

[0037]

[0038]

[0039] Through multiple verifications on the above-m...

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Abstract

The invention relates to an influenza A virus detection kit and application thereof. The kit provided by the invention contains a primer group and a probe for influenza virus detection, and specifically comprises three primers and one probe, and is a primer and probe combination designed specific for influenza A virus NP target gene. The primers and the probe disclosed by the invention are suitable for detecting influenza A virus by NASBA method, support high-throughput, rapid and accurate detection of influenza virus infection, can acquire more accurate detection result within one hour than real-time fluorescent quantitative PCR method, and have important significance for clinical application.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and relates to a kit for detecting influenza virus and an application thereof. Background technique [0002] Influenza (abbreviated as influenza) is an acute respiratory infection caused by influenza virus, which is characterized by strong infectivity and fast transmission speed. Influenza is an illness caused by the influenza virus and, as such, can spread through airborne droplets, person-to-person contact, or contact with contaminated items. Typical clinical symptoms are: sudden onset of high fever, general pain, significant fatigue and mild respiratory symptoms. Generally, autumn and winter are the high-incidence period, and the complications and death caused by it are very serious. The disease is self-limiting, but it is prone to severe complications such as pneumonia and death in infants, young children, the elderly, and patients with underlying cardiopulmonary diseases...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701
Inventor 连一霏崔江河王菡代海兵连续李荣辉鞠传余于龙魅徐明鑫梁爽
Owner MUDANJIANG MEDICAL UNIV
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