Kit for dual detection of viruses by nucleic acid and antibodies and preparation method thereof

A dual detection and kit technology, applied in the detection field, can solve the problems of high cost, high experimental conditions, and inability to be widely used, and achieve the effects of saving detection time, improving accuracy, and low cost

Active Publication Date: 2020-12-11
GUANGDONG CELL BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there are also many shortcomings, such as: high requirements for experimental conditions, high cost, and complicated analysis of later results, so it cannot be widely used in general laboratories

Method used

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  • Kit for dual detection of viruses by nucleic acid and antibodies and preparation method thereof
  • Kit for dual detection of viruses by nucleic acid and antibodies and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Design of Influenza Virus H1N1 RPA-specific Detection Primers

[0039] According to the comparison of gene sequences of common H1N1 strains, a specific conserved region was selected as the target region for primer detection. Its sequence is as follows:

[0040] 1 GTAAATTCTG TTATTGAAAA GATGAATACA CAGTTCACAG CAGTAGGTAA AGAGTTCAAC

[0041] 61 CACCTGGAAA AAAGAATAGA GAATTTAAAT AAAAAAGTTG ATGATGGTTT CCTGGACATT

[0042] 121 TGGACTTACA ATGCCGAACT GTTGGTTCTA CTGGAAAATG AAAGAACTTT GGACTACCAC

[0043] 181 GATTCAAATG TGAAGAACTT ATATGAAAAG GTAAGAAGCC AGTTAAAAAA CAATGCCAAG.

[0044] According to the rules of RPA primer design, the inventor optimized the primers, and obtained specific primer sequences through more than 300 optimization experiments, the sequences of which are shown below.

[0045] Upstream primer (SEQ ID No: 1): GTAATTCTGTTATTGAAAAGATGAATACACAGTTC

[0046] Downstream primer (SEQ ID No: 2): Biotin-CTTGGCATTGTTTTTTTAACTGGCTTCTTACCTTTTC

[0047] Probe sequ...

Embodiment 2

[0048] Example 2 Sensitivity evaluation of RPA detection method

[0049] The H1N1 virus lysate was used to extract RNA as a template, and the RPA test was carried out, and the optimal primers screened were used for RPA amplification. At the same time, ultrapure water was set as a negative control, and the reaction time (ie, the number of amplification cycles) at 40°C was 25 minutes, The RPA reaction system is 50 μl, wherein, 2 μl forward and reverse primers (10 μM), 2 μl reverse primers (10 μM), 0.6 μl probe, 25 μl containing recombinase, DNA polymerase, single-strand binding protein, endonuclease IV, Reverse transcriptase buffer, 1 μl template and 17.9 μl ddH2O, fully vortexed, mixed and transiently centrifuged, and finally added 2.5 μl of 280 mM magnesium acetate (MgOAc), and placed the reaction tube in a real-time fluorescent PCR instrument at a constant temperature of 40 ° C for a corresponding period of time; The result is as figure 1 shown.

[0050] Such as figure 1 A...

Embodiment 3

[0051] Example 3 Preparation and detection of RPA detection test strips

[0052] Prepare the detection test strip according to the conventional method, wherein the test strip has a detection line, and the biotin ligand is immobilized on the detection line; the primer described in SEQ ID NO: 2 has a biotin specific binding biotin ligand at its end. white. Specific test strips are in accordance with figure 2 form to prepare.

[0053] The test strips were used to detect H1N1 positive samples, as well as yeast, Chlamydia trachomatis, Neisseria gonorrhoeae, Staphylococcus aureus, Escherichia coli and vaginal lactobacillus samples, and the samples were amplified by RPA using primers and probes. The reaction time (i.e., the number of amplification cycles) at 40°C was 25 min, and the RPA reaction system was 50 μl, of which, 2 μl forward and reverse primers (10 μM), 2 μl reverse primers (10 μM), 0.6 μl probe, 25 μl containing Buffer for recombinase, DNA polymerase, single-strand bi...

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Abstract

The invention relates to a kit for dual detection of viruses by nucleic acid and antibodies and preparation method thereof. According to the invention, an RPA primer and a probe which specifically aimat H1N1 detection are obtained by analyzing an H1N1 sequence, and the RPA primer and the probe are prepared into a corresponding detection test strip. At the same time, monoclonal antibodies which have good effects are screened and obtained aiming at an H1N1 conserved region. An H1N1 influenza virus fluorescence quantum dot rapid detection test paper is prepared from a monoclonal antibody labeledquantum dot and other antibody labeled nitrocellulose membranes. The two detection methods are combined for use, so that the detection accuracy can be further improved, the cost is low, and the kit and method is suitable for large-scale popularization.

Description

technical field [0001] The invention relates to the detection field, in particular to a nucleic acid-antibody dual-detection virus kit and a preparation method thereof. Background technique [0002] Influenza virus (abbreviated as influenza virus) is a RNA virus that causes influenza in humans and animals. It belongs to the Orthomyxoviridae family and is divided into three types: A, B, and C. It is spherical or filamentous, with a diameter of 80-120nm. Type 3 viruses have similar biochemical and biological characteristics. The various subtypes of influenza A virus are divided according to the antigenicity of virion hemagglutinin and neuraminidase. Influenza virus causes acute upper respiratory tract infection, which spreads rapidly mainly through the patient's droplets, contact between patients or contact with contaminated objects. Influenza A virus spreads more rapidly, and the incubation period after infection is very short. , can also infect mammals and birds; the antig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844G01N33/569G01N33/577G01N33/58C07K16/10
CPCC07K16/1018C07K2317/56C12Q1/6844C12Q1/701G01N33/56983G01N33/577G01N33/588G01N2333/11C12Q2521/507C12Q2522/101
Inventor 张海涛王振
Owner GUANGDONG CELL BIOTECHNOLOGY CO LTD
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