CN101824492A

CN101824492A - Kit for detecting AH1N1 influenza virus

Info

Publication number: CN101824492A
Application number: CN 201010174472
Authority: CN
Inventors: 孙国明
Original assignee: TRUEHIGH TECH Co Ltd
Current assignee: TRUEHIGH TECH Co Ltd
Priority date: 2010-05-18
Filing date: 2010-05-18
Publication date: 2010-09-08

Abstract

The invention relates to a kit for detecting product DNA of AH1N1 influenza virus DNA amplified through a loop-mediated isothermal nucleic acid amplification technology by using gold nanoparticles, belonging to the technical field of biological detection. In combination with the loop-mediated isothermal nucleic acid amplification technology and a biological nanotechnology, the invention has the advantages that the sensitivity of biological molecular detection is improved, the operation of the method is simple, the detection is rapid, the accuracy is high, special instruments and devices are not required, and the kit can be widely used for high-sensitivity AH1N1 influenza virus detection in fields such as family diagnosis, clinical diagnosis, infectious disease control, environmental monitoring, inspection and quarantine, biotechnology and the like. The kit comprises: (1) gold nanoparticles labeled with molecular probes capable of identifying specific sequences of AH1N1 influenza viruses; and (2) a loop-mediated isothermal nucleic acid amplification system capable of amplifying AH1N1 influenza virus DNA in a sample to be detected or capable of amplifying AH1N1 influenza virus RNA in the sample to be detected after reverse transcription.

Description

A kind of test kit that detects H1N1virus

One, technical field

The present invention relates to a kind ofly detect H1N1virus DNA or RNA through the test kit of loop-mediated isothermal amplification amplification after product DNA, belong to technical field of biological with nm gold particles.

Two, background technology

To the high sensitivity of gene (DNA) and transcription product (RNA) thereof, high specific, simply, detect fast, for the early diagnosis and therapy of disease and all have crucial meaning in fields such as health and epidemic prevention, environmental monitoring, inspection and quarantine.Comparatively accurate in this respect in the past sensitive detection method has methods such as fluorescent marker method, radio-labeling, and these methods all need complicated operations and special equipment, and range of application is very narrow.The Dr.Kary B.Mullis of PE company in 1993 has invented polymerase chain reaction (Polymerase Chain Reaction), is called for short PCR.Round pcr is applied in biological scientific research and the clinical treatment on a large scale subsequently, and the detection of DNA has been entered the new epoch, and Dr.Mullis has also obtained Nobel chemistry Prize in 1993 for this reason.Though advantages such as PCR method has fast, sensitivity, accuracy is high, operation is simpler are because its reaction is carried out needing special instrument, the use of round pcr to be limited in the laboratory and hospital of specialty with the detection of product dna always.

Calendar year 2001, Japan Eiken Chemical developed a kind of Novel DNA amplification mode, loop-mediated isothermal amplification (LAMP) can be realized the efficient rapid amplifying of DNA under constant temperature, this method reaction conditions is simple, therefore DNA cloning efficient height has clear superiority aspect detection of nucleic acids.But the DNA product to LAMP amplification preparation uses conventional detection method, and as electrophoretic method, fluorescence dye method etc. all can't be got rid of the false positive problem that the LAMP amplification is brought, and need special instrument, have therefore limited the practical application of LAMP technology.

Influenza A H1N1 is the acute respiratory transmissible disease, and its pathogenic agent is a kind of novel H1N1virus, propagates in the crowd.With in the past or present seasonal influenza virus different, this virus stain includes the gene fragment of porcine influenza, bird flu and three kinds of influenza viruses of human influenza.The crowd is to the general susceptible of H1N1virus, and can the people infect the people, the early symptom that the people infects behind the first stream is similar to common influenza, comprise heating, cough, laryngalgia, physical distress, have a headache, feel cold and fatigue etc., some also can occur diarrhoea or vomiting, myalgia or tired, eyes are rubescent etc.Lack detection method simple, quick, easy to use and with low cost at H1N1virus at present.

Three, summary of the invention

The objective of the invention is to propose a kind ofly to detect the test kit of H1N1virus, thereby the existence of judging H1N1virus whether through loop-mediated isothermal amplification amplification after product DNA with nm gold particles.Test kit coupling collar mediated isothermal amplification of the present invention and biological nano technology, improve the sensitivity of biomolecule detection, it is a kind of simple to operate, quick, accurate, with low cost, method of not needing professional plant and instrument, can be widely used in home diagnostic, clinical diagnosis, transmissible disease control, environmental monitoring, inspection and quarantine, the highly sensitive influenza virus of biotechnology field detects.

Test kit of the present invention comprises two portions:

1. be marked with the nm gold particles of the molecular probe that can discern the H1N1virus particular sequence;

2. can increase in the detected sample H1N1virus DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the H1N1virus RNA in the detected sample that increases again after the reverse transcription process.

The principle of work of this test kit is:

1. nm gold particles is marked with the molecular probe that can discern the H1N1virus particular sequence, and molecular probe is fixed on the surface of nm gold particles.

2. H1N1virus dna molecular in the detected sample or H1N1virus RNA are through loop-mediated isothermal amplification massive duplication or first massive duplication after the reverse transcription process, and the target fragment of H1N1virus is exaggerated 10 ⁹Doubly, the amplified production DNA of generation has multiple tumor-necrosis factor glycoproteins.

3. mix nm gold particles and ring mediated isothermal nucleic acid amplification system, H1N1virus can be discerned the molecular probe hybridization of H1N1virus particular sequence on product D NA after the loop-mediated isothermal amplification and nm gold particles.

4. nm gold particles is along product of loop-mediated isothermal amplification dna molecular chain formation aggregate (as shown in drawings), and then cause the colour-change of nm gold particles habitat (for example in the solution, solid surface etc.), can judge according to colour-change whether testing sample has H1N1virus DNA or RNA.

Four, description of drawings

Be marked with the nm gold particles and the product D NA hybridization synoptic diagram of H1N1virus after loop-mediated isothermal amplification of molecular probe, nm gold particles is along product of loop-mediated isothermal amplification dna molecular chain formation aggregate.

Five, embodiment

Test kit of the present invention comprises 2 pipe solution, be the nm gold particles solution 50 μ l that are marked with the molecular probe that can discern the H1N1virus particular sequence in one pipe, in the pipe be 250 μ l can increase in the detected sample H1N1virus DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the H1N1virus RNA in the detected sample that increases again after the reverse transcription process.

Contain the nm gold particles that diameter is 20nm in the nm gold particles solution, concentration is 1.2nM, and the nm gold particles surface markers has the dna probe of sulfydryl modification.

Ring mediated isothermal nucleic acid amplification system composition is as follows: 0.2 μ M F3 and B3 primer, and 1.6 μ M FIP and BIP primer, 0.4Mbetaine (Sigma-Aldrich, St.Louis, MO), 10mM MgSO ₄, 1.4mM dNTPs, 1 * ThermoPol reactionbuffer (New England Biolabs, Ipswich, MA), 80 U Bst DNA polymerase (New England Biolabs), 6.25 U AMV reversed transcriptive enzyme (Invitrogen, Carlsbad, CA).

The dna probe sequence of sulfydryl modification

5′-ATGTGAAGAACTTATATAAAAAAAA-SH-3

Primer sequence

The F3 primer of H1N1virus

5′-AAGTTGATGATGGTTTCCTG-3′

The B3 primer of H1N1virus

5′-GACACTTTCCATGCACGT-3′

The FIP primer of H1N1virus

5′-CGTGGTAGTCCAAAGTTCTTTCATTCATTTGGACTTACAATGCCG-3′

The BIP primer of H1N1virus

5′-GAAGCCAGTTAAAAAACAATGCCACATTTGTGGTAAAATTCAAAGCAG-3′

Operating process is as follows

1. in the pipe that ring mediated isothermal nucleic acid amplification system is housed, add nucleic acid or the blood sample that 10 μ l extract.

2. the pipe that mixture will be housed is put into 60 ℃ water-bath 30 minutes.

3. add nm gold particles solution in the pipe in step 2 again, this moment, mixed solution was red.

4.60 ℃ water-bath 15 minutes.

The color of solution in the pipe behind the observing response, solution keep red constant influenza infection specific RNA or the H1N1virus DNA of then not containing; If solution becomes lilac, then judge and contain specific RNA of influenza infection or H1N1virus DNA in the sample.In such cases, if sample is a blood sample, provide the people of blood sample to be infected so by H1N1virus.

Sequence table

＜110〉Truehigh Tech Co., Ltd.

＜120〉a kind of test kit that detects H1N1virus

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Claims

1. one kind is detected the test kit of H1N1virus through loop-mediated isothermal amplification amplification after product DNA with nm gold particles, and it is characterized in that this test kit comprises with the lower section: (1) is marked with the nm gold particles of the molecular probe that can discern the H1N1virus particular sequence; (2) can increase in the detected sample H1N1virus DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the H1N1virus RNA in the detected sample that increases again after the reverse transcription process.

2. according to claim 1, the principle of work of this test kit be (1) with the H1N1virus DNA in the loop-mediated isothermal amplification amplification detected sample or earlier through after the reverse transcription process with the H1N1virus RNA in the loop-mediated isothermal amplification amplification detected sample; (2) nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization; (3) hybridization causes the nm gold particles gathering, causes nm gold particles habitat color change, judges with this whether detected sample contains H1N1virus DNA or RNA.

3. according to claim 1, the nm gold particles probe comprises two parts, the one, and the gold grain of nanoscale, another part are the molecular probes that is fixed on the nm gold particles surface, this probe can be discerned the particular sequence of H1N1virus.

4. according to claim 2, the amplification object of loop-mediated isothermal amplification can be the mixture of H1N1virus DNA, RNA or DNA and RNA in the detected sample.

5. according to claim 2, nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization can be carried out under same reaction conditions, in the same reaction container simultaneously with the ring mediated isothermal nucleic acid amplification reaction.

6. according to claim 2, nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization can occur in the solution or solid surface.

7. according to claim 2, nm gold particles probe and product of loop-mediated isothermal amplification DNA hybridization cause solution or solid surface color change, and this variation can directly be judged by naked eyes, also can utilize opticinstrument detections such as spectrophotometer.

8. according to claim 3, molecular probe be meant can identification ring mediated isothermal amplification product D NA functional molecular, these molecules comprise DNA, RNA, PNA, protein, chemical molecular.

9. according to claim 4, same reaction conditions includes but not limited to: solution composition, constituent concentration, pH value, temperature, reaction times etc.