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RDA method and kit for rapidly detecting influenza A virus

An influenza A virus and kit technology, applied in the field of molecular biology, can solve the problems of lack of influenza A detection kits, difficulty in obtaining, hindering rapid qualitative detection of influenza, etc., and achieve low detection cost, low mass production cost, The effect of high-precision rapid molecular detection

Pending Publication Date: 2020-05-08
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the traditional influenza nucleic acid detection methods are based on PCR. The detection needs to rely on PCR machines or expensive real-time quantitative PCR machines. Multiple operations are required, including virus lysis, viral RNA extraction and molecular detection. The entire process is usually concentrated in the laboratory. In-house, requires sophisticated instruments and skilled operators
However, these conditions are often difficult to obtain in grassroots community clinics, which seriously hinders the rapid qualitative detection of influenza.
At present, there is still a lack of simple, cheap and highly automated influenza A detection kits suitable for grassroots inspections

Method used

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  • RDA method and kit for rapidly detecting influenza A virus
  • RDA method and kit for rapidly detecting influenza A virus
  • RDA method and kit for rapidly detecting influenza A virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Establishment of a detection method for influenza A virus (FluA) RDA fluorescence detection kit

[0082] (1) Acquisition of recombinant enzyme KX and KY proteins

[0083] The reported recombinant enzyme UvsX has poor stability and is difficult to mass produce and store for a long time. In order to solve this problem, the research and development team used bioinformatics methods to analyze and simulate large quantities of protein structures, and finally found a new recombinant enzyme Enzyme KX and its accessory protein KY.

[0084] In this example, the R&D team extracted the key functional site information in the recombinase structure, such as DNA binding site, ATP hydrolysis site, etc., and mapped it to the three-dimensional structure of the protein to obtain secondary structure information and tertiary structure information , by integrating the functional residues of the primary structure sequence, secondary structure features and tertiary structure spatial ...

Embodiment 2

[0135] Example 2 RDA fluorescence method detection reagent sensitivity test

[0136] The positive control is the pUC57-M1 plasmid containing the M1 gene of influenza A virus (FluA), and the negative control is the empty vector pUC57 plasmid.

[0137] The specific operation is as follows:

[0138] Step 1: Dilute the positive control plasmid to 10^4c, and then dilute to 10^3c, 10^2c, and 10^1c by 10-fold serial dilution.

[0139] Step two, sample processing. Take 5 μL of each concentration of the plasmid in step 1 in an EP tube, and take 5 μL of the negative control in another EP tube, add 20 μL of Buffer A respectively, shake and mix, and let stand at room temperature for 10-15 minutes;

[0140] Step 3, system preparation and testing. Add 25 μL Buffer B to each tube, shake and mix, add 50 μL of the mixed solution to the RDA fluorescence method reaction module, cover the tube cap, shake and centrifuge, and detect immediately; the reaction program is: 39°C for 1 minute, 30 cyc...

Embodiment 3

[0152] Example 3 RDA fluorescence method detection reagent specificity test

[0153] 3 cases of influenza A virus (Influenza A virus, FluA), 1 case of influenza B virus (Influenza B virus, FluB), and 1 case of respiratory syncytial virus (Respiratory Sycytial Virus, RSV) were collected clinically, a total of 5 cases It was verified by fluorescent quantitative PCR that the corresponding virus-positive samples were tested to test the specificity of the kit.

[0154] The specific operation is as follows:

[0155] Step 1. Sample processing. Take 5 μL of each of the above 5 positive samples in EP tubes, and at the same time take 5 μL of the positive control and negative control of the kit in a new EP tube, add 20 μL Buffer A respectively, shake and mix, and let stand at room temperature for 10-15 minutes;

[0156] Step 3, system preparation and testing. Add 25 μL Buffer B to each tube, shake and mix, add 50 μL of the mixed solution to the RDA fluorescence method reaction module,...

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Abstract

The invention discloses an RDA method and a kit for rapidly detecting influenza A virus. The kit comprises a specific primer group and an RDA fluorescence labeling probe so as to realize safe, specific, sensitive, simple and convenient detection of the influenza A virus (FluA), and to make up for the defects of existing traditional detection technologies. According to the kit provided by the invention, a nucleic acid extraction step can be omitted, the influenza A virus can be detected within 20 minutes at a constant temperature of 37-42 DEG C, the specificity is 100%, and the kit is very suitable for on-site rapid detection. Compared with common PCR methods, the RDA fluorescence method has the advantages that: the reaction is carried out at constant temperature, the temperature change isnot needed, complex instruments are not needed, and the reaction time is short. Therefore, the method and the kit provided by the invention have the characteristics of simple and rapid operation, goodspecificity, high sensitivity, low cost and the like, provide an effective a technical means for on-site rapid detection and screening of the influenza A virus, and have a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a primer, a probe and a related kit for detecting influenza A virus nucleic acid based on RDA fluorescence detection technology. Background technique [0002] Influenza virus belongs to the Orthomyxoviridae family and is a single-stranded RNA membrane virus. It is the most common respiratory virus and is divided into three types: A (A), B (B) and C (C). Influenza A and B are the most common types of influenza virus, and they occur almost once a year in China. Among them, influenza A virus is the most likely to mutate and is highly pathogenic to humans. It has caused worldwide pandemics many times. The structure of influenza A virus can be divided into three parts: envelope, matrix protein and core from the outside to the inside. There are two types of surface glycoproteins on the outer granule membrane. The first type is hemagglutinin (H), which is divi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2563/107Y02A50/30
Inventor 刘华勇谢婵芳季宇陈翀
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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