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RDA method and kit for rapidly detecting respiratory syncytial virus

A technology for syncytial virus and respiratory tract, applied in the field of molecular biology, can solve the problems of lack of RSV detection kits, difficult to obtain, hindering rapid diagnosis of influenza, etc., and achieve high-precision rapid molecular detection, low mass production cost, and low detection cost. Effect

Pending Publication Date: 2021-02-02
GUANGZHOU PLUSLIFE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • RDA method and kit for rapidly detecting respiratory syncytial virus
  • RDA method and kit for rapidly detecting respiratory syncytial virus
  • RDA method and kit for rapidly detecting respiratory syncytial virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 A Respiratory Syncytial Virus (RSV) RDA Fluorescence Detection Kit

[0083] (1) Acquisition of recombinant enzyme KX and KY proteins

[0084] 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.

[0085] 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 distance, a data model for pr...

Embodiment 2

[0136] Example 2 RDA fluorescence method detection reagent sensitivity test

[0137] The positive control is the pUC57-L plasmid containing the L gene of respiratory syncytial virus (RSV), and the negative control is the empty vector pUC57 plasmid.

[0138] The specific operation is as follows:

[0139] 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.

[0140] 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;

[0141] Step 3, 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 reaction module, cover the tube cap, shake and centrifuge, and detect immediately; the reaction program is: 39°C for 1 minute, 30 ...

Embodiment 3

[0153] Example 3 RDA fluorescence method detection reagent specificity test

[0154] 2 cases of respiratory syncytial virus (Respiratory Sycytial Virus, RSV), 1 case of influenza A virus (Influenza A virus, FluA), and 1 case of influenza B virus (Influenza B virus, FluB) were collected clinically, a total of 4 cases It was verified by fluorescent quantitative PCR that the corresponding virus-positive samples were tested to test the specificity of the kit.

[0155] The specific operation is as follows:

[0156] Step 1. Sample processing. Take 5 μL of each of the above 4 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;

[0157] Step 3, system preparation and testing. Add 25 μL of Buffer B to each tube, shake and mix, add 50 μL of the mixture to the RDA fluorescence method reaction module, cov...

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Abstract

The invention discloses an RDA method and kit for rapidly detecting respiratory syncytial viruses. The kit comprise a specific primer pair and an RDA fluorescence labeling probe to realize safe, specific, sensitive, simple and convenient detection of the respiratory syncytial viruses (RSV) and overcome the defects of the existing traditional detection technology. According to the kit provided by the invention, a nucleic acid extraction step can be omitted, the respiratory syncytial viruses can be detected at the constant temperature of 37-42 DEG C within 20 minutes, the specificity is 100%, and the kit is very suitable for on-site rapid detection. Compared with a common PCR method, the RDA fluorescence method has the advantages that the reaction is performed at constant temperature, the temperature change is not needed, complex instruments are not needed, and the reaction time is short. Therefore, the method and the kit have the characteristics of simplicity and quickness in operation,good specificity, high sensitivity, low cost and the like, provide an effective technical means for on-site quick detection and screening of the respiratory syncytial viruses, 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 pair, a probe and a related kit for detecting respiratory syncytial virus nucleic acid based on RDA fluorescence detection technology. Background technique [0002] Respiratory syncytial virus (Respiratory Sycytial Virus, RSV) belongs to the Paramyxoviridae Pneumovirus genus, is an enveloped non-segmented single-stranded RNA (negative strand) virus. Its genome consists of about 15kb, encoding 10 genes, among which the F gene encoding the fusion protein is relatively conservative. RSV can cause cell fusion lesions in cells. According to this feature, it is named respiratory syncytial virus. Humans are its only natural host, and it mainly infects human nasopharyngeal epithelial cells. It is the most common virus that causes lower respiratory tract infections in infants and young children. It invades the human body mainly through the respiratory ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/507C12Q2537/1376C12Q2563/107C12Q2527/125C12Q2545/113Y02A50/30
Inventor 谢婵芳刘华勇黄嘉恩陈翀
Owner GUANGZHOU PLUSLIFE BIOTECH CO LTD
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