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-hous

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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

Example Embodiment

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

[0082] (1) Acquisition of recombinase KX and KY protein

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

[0084] In this example, the R&D team extracts key functional site information in the recombinase structure, such as DNA binding sites, ATP hydrolysis sites, etc., and maps them 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, the secondary structure characteristics and the spatial distance of the tertiary structure, a data model for recom...

Example Embodiment

[0135] Embodiment 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 operations are as follows:

[0138] Step 1. Dilute the positive control plasmid to 10^4c, and then dilute the positive control plasmid to 10^3c, 10^2c, and 10^1c.

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

[0140] Step three, system preparation and testing. Add 25μL of Buffer B to each tube, shake and mix, add 50μL of the mixed solution to the RDA fluorescence method reaction module, cover the tube cap, shake and centrifuge for immediate detection; the reaction procedure is: 39℃ for 1 minute, ...

Example Embodiment

[0152] Example 3 RDA fluorescence detection reagent specificity test

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

[0154] The specific operations are as follows:

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

[0156] Step three, system preparation and testing. Add 25 μL of 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 and shake and centrifuge for imme...

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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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