Kit and method for detecting influenza virus based on immune magnetic bead enrichment

A detection kit, influenza virus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as reducing the detection rate of weakly positive clinical specimens, and achieve low hardware requirements, high sensitivity, and simple operation. Effect

Inactive Publication Date: 2013-10-30
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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AI Technical Summary

Problems solved by technology

However, all current nucleic acid detection methods require the use of commercial kits to purify the nucleic acids in clinical samples before in vitro amplification, otherwise false negatives are likely to occur and the detection rate of weakly positive clinical samples will be reduced

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A detection kit for influenza A (H1N1) virus, using this kit to detect 96 clinical specimens of nasopharyngeal swabs (80 positive specimens, 15 negative specimens, and 1 blank specimen), the detection process took 1 hour, The accuracy rate is 100%.

[0031] This kit includes an immune enrichment reaction system for specifically enriching and detecting influenza A (H1N1) virus in a sample; a loop-mediated isothermal nucleic acid amplification system for amplifying and detecting RNA of influenza A (H1N1) virus in a sample, as follows :

[0032] A. Components of immune enrichment reaction system: Magnetic beads labeled with influenza A H1N1 virus-specific monoclonal antibody (diameter: 100-300 nm; working dilution concentration: 1:10-1:20); 0.1 M phosphate buffer solution (PBS) pH 7.2- pH 7.4; sterile ultrapure water

[0033]B. Loop-mediated isothermal nucleic acid amplification system components: Bst DNA polymerase (8 U / μl, New England Biolabs), 10×Bst DNA polymerase bu...

Embodiment 2

[0053] A detection method for influenza A (H1N1) virus, the steps are:

[0054] A. Antibody preparation: use standard B lymphocyte hybridoma technology [Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497] to prepare H1N1 virus-specific monoclonal antibody , and the prepared antibody was purified by n-caprylic acid-saturated ammonium sulfate precipitation [Perosa F, Carbone R, Ferrone S, Dammacco F (1990) Purification of human imunoglobulins by sequential precipitation with caprylic acid and ammonium sulphate. J Immunol Methods 128 : 9–16.]

[0055] B. Preparation of immunomagnetic beads: wash the magnetic beads with 50 mM sodium acetate buffer at pH 4.7 containing 0.1% (v / v) Tween20, add EDC and NHS to make the final concentration 20 mM, and react at room temperature for one hour , wash the magnetic beads, add H1N1 virus-specific monoclonal antibody so that the ratio of the number of carboxyl groups...

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Abstract

The invention discloses a kit and a method for detecting influenza A virus based on immune magnetic bead enrichment. The kit comprises A, an immune enrichment reaction system component and B, a loop-mediated isothermal nucleic acid amplification system component; and the preparation method of the components of the kit comprises C, preparing an antibody, D, preparing immune magnetic beads and E, designing a specific primer of influenza A virus subtype H1N1 for loop-mediated isothermal amplification reaction at 65 DEG C. The detection method comprises the following steps of: a, performing immune enrichment; b, thermally cracking a product, separating through a magnetic frame and taking supernate containing virus nucleic acid as a template for the loop-medicated isothermal amplification reaction; c, performing the loop-medicated isothermal amplification reaction; and d, determining a result. The kit has the advantages of high specificity, high sensitivity, low hardware requirement and the like, is simply and quickly operated, and can be widely applied to the detection and monitoring of the influenza A virus of samples, such as nasopharyngeal swabs and the like, of fever patients in all grades of disease prevention and control mechanisms, influenza monitoring network laboratories, sentinel point hospitals and the like.

Description

technical field [0001] The invention belongs to the application field of biotechnology, more specifically relates to a detection kit for influenza A (H1N1) virus, and also relates to a detection method for influenza A (H1N1) virus, which can be applied to disease prevention and control institutions at all levels and influenza monitoring networks Laboratory, sentinel hospital, etc. detect and monitor influenza A H1N1 influenza virus in samples such as nasopharyngeal swabs of fever patients. Background technique [0002] In March 2009, a "human-infected swine flu" epidemic broke out in Mexico, resulting in deaths. The study found that the pathogen of this outbreak was a mutated new type A H1N1 virus, including gene fragments of three influenza viruses: swine flu, bird flu and human flu. The virus is highly contagious and can be transmitted through close-range droplets and contact. The main clinical manifestations are flu-like symptoms, a few cases are severe and progress rap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70
Inventor 张先恩王殿冰危宏平张治平
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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