CN101812544A

CN101812544A - Influenza virus detection kit

Info

Publication number: CN101812544A
Application number: CN 201010164055
Authority: CN
Inventors: 孙国明
Original assignee: TRUEHIGH TECH Co Ltd
Current assignee: TRUEHIGH TECH Co Ltd
Priority date: 2010-05-06
Filing date: 2010-05-06
Publication date: 2010-08-25

Abstract

The invention relates to a kit for detecting product DNA by utilizing nano-gold particles after influenza virus is amplified through a loop-mediated isothermal nucleic acid amplification technique, which belongs to the technical field of biological detection. The kit combines the loop-mediated isothermal nucleic acid amplification technique with a biological nanotechnology to improve the detection sensitivity of biological molecules, is a method with simple operation, rapidness, accuracy and no need for professional instruments and equipment, and can be widely applied to high-sensitivity influenza virus detection in the fields of household diagnosis, clinical diagnosis, infectious diseases control, environment monitoring, inspection and quarantine, biotechnology and the like. The kit comprises two parts, namely (1) the nano-gold particles marked with molecular robes capable of identifying specific sequences of influenza virus M1 and (2) a loop-mediated isothermal nucleic acid amplification system capable of amplifying the escherichia coli DNA in to-be-detected samples or performing a reverse transcription process and then amplifying the RNA of influenza virus M1 gene in the to-be-detected samples.

Description

A kind of test kit that detects influenza virus

One, technical field

The present invention relates to a kind ofly detect influenza virus M1 gene 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 virus; be called for short influenza virus; be that a kind of mankind of causing and animal suffer from grippal RNA viruses; on taxonomy; influenza virus belongs to Orthomyxoviridae family; it can cause acute upper respiratory tract infection, and propagates rapidly by air, and regular meeting has periodically and is very popular all over the world.Influenza virus can cause more serious symptom the more weak old man of immunizing power or the patient of child and some immune disorders, as pneumonia or cardiopulmonary depletion etc.Lack detection method simple, quick, easy to use and with low cost at influenza virus at present.

The M1 stromatin is made up of more than 200 amino acid, the about 26KDa of molecular weight, and it is the primary structure albumen of virus, accounts for 30%～40% of influenza virus protein total amount.This albumen has type specificity, and its antigenic specificity is one of foundation of influenza virus somatotype, and can judge the existence of influenza virus according to the existence of M1 gene.

Three, summary of the invention

The objective of the invention is to propose a kind ofly to detect the test kit of influenza virus M1 gene, thereby the existence of judging influenza virus 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 influenza virus M1 gene particular sequence;

2. can increase in the detected sample influenza virus M1 gene DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the influenza virus M1 gene 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 influenza virus M1 gene particular sequence, and molecular probe is fixed on the surface of nm gold particles.

2. influenza virus M1 gene DNA molecule in the detected sample or influenza virus M1 gene RNA are through loop-mediated isothermal amplification massive duplication or first massive duplication after the reverse transcription process, and the target fragment of influenza virus M1 gene 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, influenza virus M1 gene can be discerned the molecular probe hybridization of influenza virus M1 gene 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 influenza virus M1 gene DNA or RNA.

Four, description of drawings

Be marked with the nm gold particles and the product D NA hybridization synoptic diagram of influenza virus M1 gene 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 influenza virus M1 gene particular sequence in one pipe, in the pipe be 250 μ l can increase in the detected sample influenza virus M1 gene DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the influenza virus M1 gene 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, 1.6 μ M FIP and BIP primer, 0.8 μ M LoopF and LoopB primer, 0.4M betaine (Sigma-Aldrich, St.Louis, MO), 10mM MgSO4,1.4mM dNTPs, 1 * ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 80U Bst DNApolymerase (New England Biolabs) .6.25U AMV reversed transcriptive enzyme (Invitrogen, Carlsbad, CA).

The dna probe sequence of sulfydryl modification

5′-AGTGGCACCAGGCCAGAAAAAAAA-SH-3′

Primer sequence

The dna probe sequence of sulfydryl modification

5′-GATCGCACAGAGACTTAAAAAAAA-SH-3′

Primer sequence

The F3 primer of M1

5′-GAGTCTTCTAACCGAGGTCGAAACG-3′

The B3 primer of M1

5′-TTCCCATTGAGGGCATTTTGGAC-3′

The BIP primer of M1

5′-CCTTAGTCAGAGGTGACAGGTACGATGCAGTCCTCGCTCAGTGGG-3’

The FIP primer of M1

5 '-TGTCTTTAGCCATTCCATGAGTCAGGCCCCCTCAAAGCCGA-3 ' M1's

The LoopF primer of M1

5’-GTTCTTTCCAGCAAAGACATC-3’

The LoopB primer of M1

5’-GTGTTCACGCTCACCG-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.

5. the color of solution in the pipe behind the observing response, solution keeps red constant specific M1 gene RNA of influenza infection or the influenza virus M1 gene DNA of then not containing; If solution becomes lilac, then judge and contain specific M1 gene RNA of influenza infection or influenza virus M1 gene DNA in the sample.In such cases, if sample is a blood sample, provide the people of blood sample to be infected so by influenza virus.

Sequence table

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＜120〉a kind of test kit that detects influenza virus

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Claims

1. one kind is detected the test kit of influenza virus M1 gene 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 influenza virus M1 gene particular sequence; (2) can increase in the detected sample influenza virus M1 gene DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the influenza virus M1 gene 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 influenza virus M1 gene DNA in the loop-mediated isothermal amplification amplification detected sample or earlier through after the reverse transcription process with the influenza virus M1 gene 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 influenza virus M1 gene 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 influenza virus M1 gene.

4. according to claim 2, the amplification object of loop-mediated isothermal amplification can be the mixture of influenza virus M1 gene 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.