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Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses

An influenza virus, ring-mediated constant temperature technology, applied in the biological field, can solve the problems of poor specificity, time-consuming and laborious, etc., and achieve the effect of rapid detection and high specificity

Inactive Publication Date: 2011-06-29
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

[0005] Influenza A virus is an RNA virus. Traditional genetic detection techniques include Northern hybridization, Southern hybridization, Western blotting, PCR, etc. These detection methods are not only time-consuming and laborious, but also have poor specificity.
In the past ten years, real-time fluorescence quantitative PCR has developed into an important gene detection technology, which has high sensitivity and specificity, but requires expensive instruments and professional operation

Method used

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  • Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses
  • Loop-mediated isothermal amplification detection method and kit for H1N1 influenza A viruses

Examples

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Embodiment 1

[0035] Example 1: Design of LAMP primers and probes for influenza A (H1N1) virus

[0036] Find the PA gene fragment sequences of all influenza A viruses in NCBI http: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / request.cgi, and find out the sequences of the influenza A viruses through multiple comparisons Conserved segment. Primers and probes were designed on the conserved fragments using PrimerExplorer.

[0037] PrimerExplorer was used to design primers and probes for the PA gene fragment sequence of influenza A H1N1 (2009 epidemic). Compare the designed primers and probes with all viral sequences to find the most variable primers and probes.

[0038] The full-length PA gene sequence of the above-mentioned influenza A H1N1 (popular in 2009) was obtained from NCBI (>gi|238914637|gb|GQ244322.1|Influenza A virus (A / Guangdong / 05 / 2009(H1N1)) segment 3 polymerase PA( PA)gene,completecds)

[0039] The design result is:

[0040] Primers and specific probes for Influenza A (H1N1) ...

Embodiment 2

[0051] Embodiment two: the preparation of the nucleic acid nano-gold biosensor for detecting influenza A virus according to the present invention

[0052] 1. Preparation of nano gold (colloidal gold):

[0053] Add 100ml of 0.01% HAuCL to a 500ML round bottom flask 4 solution, heated to boiling while stirring; add 2ml of 1% sodium citrate to the above solution, the solution turns blue within 20s, turns wine red after 60s, continues to boil for 10min, stops heating and continues stirring for 15min; colloidal gold solution 4 Stored in the dark at ℃, gold nanoparticles are identified by the maximum absorbance value at 520nm.

[0054] 2. Preparation of gold-labeled oligonucleotide probes: Dissolve 1OD DNA-probe 1 in 100 μl deionized water, add to 5-fold volume concentrated colloidal gold solution, keep at 4°C for 24 hours; 10% bovine serum albumin After blocking for 30 minutes, add NaCl and 1% SDS to final concentrations of 0.1M and 0.01%, respectively, overnight at 4°C, centrifu...

Embodiment 3

[0065] Embodiment 3: Preparation and detection method of the kit for detecting the nucleic acid nano-gold biosensor of influenza A (H1N1) virus according to the present invention

[0066] The kit of A H1N1 influenza virus nucleic acid nano-gold biosensor includes the following components:

[0067] LAMP Reagent, Influenza A H1N1 Influenza Virus Nucleic Acid Nanogold Biosensor;

[0068]The LAMP reaction reagents described therein include: 10 times RT-PCR buffer 150 μl, dNTP solution 150 μl, magnesium ions 150 μl, enzyme mixture 60 μl, RNase inhibitor 30 μl, upstream outer primer 30 μl, downstream outer primer 30 μl, upstream inner primer 30 μl, downstream inner primer 30 μl; wherein, the upstream outer primer contains the nucleotide sequence shown in SEQ ID NO.1, the downstream outer primer contains the nucleotide sequence shown in SEQ ID NO.3, and the upstream inner primer contains the nucleotide sequence shown in SEQ ID NO.3. The nucleotide sequence shown in SEQ ID NO.2, the ...

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Abstract

The invention relates to a method for detecting H1N1 influenza A viruses based on the LAMP (loop-mediated isothermal amplification of nucleic acid) technology, which comprises the following steps: extracting virus RNA (ribonucleic acid); carrying out reverse transcription and LAMP amplification; converting the LAMP amplification product into single-chain substances; and detecting the LAMP amplification product by using a nucleic acid nano-gold biosensor. The invention also provides a kit for detecting H1N1 influenza A viruses. By using the specially-designed LAMP inner primer to introduce an enzyme cutting site sequence segment into the amplification product in the LAMP amplification process, using the restriction endoenzyme TspR I to digest the amplification product and acquiring a large number of single-chain DNA (deoxyribonucleic acid) products with the length of 140bp or so by physical shock cooling, the combination of the LAMP technology based on the specific primer and the nucleic acid nano-gold biosensor with a specific probe ensures that the detection method and kit has high specificity and high detection speed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for rapid detection of influenza A (H1N1) virus nucleic acid and a detection product. Background technique [0002] Influenza, referred to as influenza, is an acute respiratory infectious disease caused by three types of influenza viruses: A, B, and C. Among them, the host of influenza A virus is the most extensive and the most harmful, and humans, poultry, and livestock are all hosts of influenza A virus. According to its surface structure (hemagglutinin H and neuraminidase N) and its genetic characteristics, influenza A virus can be divided into many subtypes. Up to now, influenza A virus has discovered 16 subtypes of hemagglutinin (H1-H16), neuraminidase has 9 subtypes (N1-N9). [0003] In 2009, the influenza A (H1N1) influenza epidemic, which broke out from Mexico and spread to many countries around the world, was caused by a new variant of the influenza A (H1N1) virus, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 曾令文刘杰顿博影赵昌路
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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