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Nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses

An influenza A virus and biosensor technology, applied in the biological field, can solve the problems of time-consuming, labor-intensive, non-productized, complex and expensive transmission electron microscopy, etc.

Inactive Publication Date: 2011-01-12
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned detection technologies have their own shortcomings. For example, Mirkin's technology requires a complex and expensive transmission electron microscope, Wang needs expensive imported slides, and requires a long time of pre-hybridization and hybridization washing, and Xi Dong's detection requires a long time. Complicated pre-hybridization, hybridization, membrane washing and silver staining process, no technical advantages, time-consuming and labor-intensive, and they are all in the stage of experimental research, no commercialization

Method used

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  • Nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses
  • Nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses
  • Nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: Design of specific primers and probes for influenza A virus and influenza A H1N1 virus

[0052] At NCBI http: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / request.cgi Find out all the MP gene fragment sequences of influenza A virus in, and find the conservative segment of influenza A virus through multiple alignment. Express Primer is used to design primers and probes on its conservative fragments.

[0053] The primers and probes were designed by using Express Primer based on the sequence of the HA gene fragment of H1N1 (2009 epidemic). Align the designed primers and probes with all viral sequences to find the primers and probes with the strongest variability.

[0054] The full-length HA gene sequence of the above type A H1N1 (popular in 2009) is derived from NCBI (>gi|227809829|gb|FJ966082.1|InfluenzaAvirus(A / California / 04 / 2009(H1N1)) segment 4 hemagglutinin(HA)gene , Complete cds)

[0055] The design results are:

[0056] Specific probes and primers for influenza ...

Embodiment 2

[0069] Example 2: Preparation of the nucleic acid nano-gold biosensor for detecting influenza A virus according to the present invention

[0070] 1. Preparation of nano gold (colloidal gold):

[0071] Add 100ml 0.01% HAuCL to a 500ML round bottom flask 4 The solution is heated to boiling while stirring; 2ml of 1% sodium citrate is added to the above solution, the solution turns blue within 20 seconds, and turns to wine red after 60 seconds. Continue boiling for 10 minutes, stop heating and continue stirring for 15 minutes; colloidal gold solution Store in the dark at 4°C, and the nano-gold is identified by the maximum absorbance value at 520nm.

[0072] 2. Preparation of gold-labeled oligonucleotide probe: Dissolve 1OD DNA-probe 2 in 100μl deionized water, add it to 5 times the volume of concentrated colloidal gold solution, 4℃ for 24 hours; 10% bovine serum albumin After blocking for 30 minutes, add NaCl and 1% SDS to the final concentration of 0.1M and 0.01%, respectively, overnig...

Embodiment 3

[0083] Example 3: Preparation and detection method of the kit for detecting influenza A virus and influenza A H1N1 nucleic acid nano-gold biosensor according to the present invention

[0084] The kit of influenza A virus and influenza A H1N1 influenza virus nucleic acid nano-gold biosensor includes the following components:

[0085] RT-asymmetric PCR reaction reagent, influenza A virus and influenza A H1N1 virus nucleic acid nano-gold biosensor;

[0086] The RT-asymmetric PCR reaction reagents include: 10 times RT-PCR buffer 150μl, dNTP solution 150μl, magnesium ion 150μl, enzyme mixture 60μl, RNase inhibitor 30μl, type A upstream primer 30μl, type A downstream Primer 30μl, H1N1 upstream primer 30μl, H1N1 downstream primer 30μl; among them, the upstream primer used to detect influenza A virus contains the nucleotide sequence shown in SEQ ID NO. 4, and the downstream primer contains the nucleotide sequence shown in SEQ ID. The nucleotide sequence shown in NO.5; the upstream primer fo...

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Abstract

The invention relates to a nucleic acid nano-gold biosensor for detecting influenza A viruses and influenza A (H1N1) viruses. The nucleic acid nano-gold biosensor comprises a sample pad, glass fiber, a nitrocellulose membrane and absorbent paper fixed on a rubber plate in turn from left to right; the glass fiber is coated with nano-gold marked oligonucleotide probes which have a nucleotide sequence shown by SEQ ID No.2 or 7; two kinds of oligonucleotide probes are immobilized on the nitrocellulose membrane; the nitrocellulose membrane probes immobilized close to one end of the absorbent paper form a quality control line and have a nucleotide sequence shown by SEQ ID No.3 or 8; and the nitrocellulose membrane probes immobilized close to one end of the glass fiber form a detection line and have a nucleotide sequence shown by SEQ ID No.1 or 6. The nucleic acid nano-gold biosensor has the advantages of simple manufacturing and fast detection without needing professional technicians and expensive instrument and equipment.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a rapid detection method and detection product for the nucleic acid of influenza A virus and influenza A H1N1 virus. Background technique [0002] Influenza is abbreviated as influenza, which is an acute respiratory infectious disease caused by three influenza viruses of type A, type B and type C. Among them, influenza A viruses have the widest and most harmful hosts. Humans, poultry, and livestock are all hosts of influenza A viruses. According to its surface structure (hemagglutinin H and neuraminidase N) and its genetic characteristics, influenza A viruses can be divided into many subtypes. So far, influenza A viruses have found 16 subtypes of hemagglutinin (H1-H16), neuraminidase has 9 subtypes (N1-N9). [0003] The human infection with influenza A (H1N1) that broke out in Mexico in 2009 and spread to many countries around the world was caused by a newly mutated influenza A (H1N1) virus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/34C12Q1/70C12Q1/68C12R1/93
Inventor 曾令文顿博影刘国东
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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