CN102226222A - Kit for detecting coxsackie virus A16 - Google Patents
Kit for detecting coxsackie virus A16 Download PDFInfo
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- CN102226222A CN102226222A CN2011101557911A CN201110155791A CN102226222A CN 102226222 A CN102226222 A CN 102226222A CN 2011101557911 A CN2011101557911 A CN 2011101557911A CN 201110155791 A CN201110155791 A CN 201110155791A CN 102226222 A CN102226222 A CN 102226222A
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Abstract
The invention relates to a kit for detecting product deoxyribonucleic acid (DNA) which is amplified from coxsackie virus A16 subjected to a loop-mediated isothermal amplification technology by using nano-gold particles, belonging to the technical field of biological detection. By combining the loop-mediated isothermal amplification technology and biological nanometer technology, the sensitivity of biological molecular detection is improved, so the invention is a method with simplicity in operation, quickness and accuracy without professional instrument and equipment, and the kit can be widely applied to high sensitivity coxsackie virus A16 detection in the fields of household diagnosis, clinical diagnosis, infectious disease control, environmental monitoring, inspection and quarantine, biological technology and the like. The kit comprises two parts: (1) nano-gold particles marked with molecular probes which can identify the specific sequence of the coxsackie virus A16; and (2) a loop-mediated isothermal amplification system which can amplify the DNA of the coxsackie virus A16 in a sample to be detected, or amplify ribonucleic acid (RNA) of the coxsackie virus A16 in the sample to be detected after a reverse transcription process.
Description
Technical field
The present invention relates to a kind ofly detect coxsackie virus A 16-type 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.
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.
Coxsackie virus (Coxsachie virus) is that a kind of enterovirus is divided into A and B two classes, and Coxsackie virus is the common virus through respiratory tract and digestive tract infection human body of a class, infects cold symptoms such as people from back can occur generating heat, sneezes, cough.16 types of CA group abbreviate coxsackie virus A 16-type (COX A16) as, are the main virus that causes hand foot mouth disease.Small rice grain or mung bean size, rubescent canescence exanthema vesiculosum or red papule on every side appear in position such as cheek, tongue, soft palate, hard palate, lip inboard, brothers' heart, elbow, knee, buttocks and external genitalia in the infant oral cavity of being invaded by coxsackie virus A 16-type.Lack detection method simple, quick, easy to use and with low cost at coxsackie virus A 16-type at present.
Summary of the invention
The objective of the invention is to propose a kind ofly to detect the test kit of the proprietary gene fragment of coxsackie virus A 16-type, thereby the existence of judging coxsackie virus A 16-type whether through loop-mediated isothermal amplification amplification after product DNA with nm gold particles.
The present invention adopts following employing following technical proposals to solve the problems referred to above: test kit of the present invention comprises two portions: (1) is marked with the nm gold particles of the molecular probe that can discern the coxsackie virus A 16-type particular sequence; (2) can increase in the detected sample coxsackie virus A 16-type DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the coxsackie virus A 16-type RNA in the detected sample that increases again after the reverse transcription process.
Among the present invention, the principle of work of test kit is: 1. nm gold particles is marked with the molecular probe that can discern the coxsackie virus A 16-type particular sequence, and molecular probe is fixed on the surface of nm gold particles.
2. coxsackie virus A 16-type dna molecular in the detected sample or coxsackie virus A 16-type RNA are through loop-mediated isothermal amplification massive duplication or first massive duplication after the reverse transcription process, and the target fragment of coxsackie virus A 16-type 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, coxsackie virus A 16-type can be discerned the molecular probe hybridization of coxsackie virus A 16-type 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 coxsackie virus A 16-type DNA or RNA.
Among the present invention, 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 coxsackie virus A 16-type.
Among the present invention, the amplification object of loop-mediated isothermal amplification can be the mixture of coxsackie virus A 16-type DNA, RNA or DNA and RNA in the detected sample.
Among the present invention, nm gold particles probe and coxsackie virus A 16-type 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.
Among the present invention, nm gold particles probe and coxsackie virus A 16-type product of loop-mediated isothermal amplification DNA hybridization can occur in the solution or solid surface.
Among the present invention, nm gold particles probe and coxsackie virus A 16-type 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.
Among the present invention, molecular probe is meant the functional molecular that can discern coxsackie virus A 16-type product of loop-mediated isothermal amplification DNA, and these molecules comprise DNA, RNA, PNA, protein, chemical molecular.
Among the present invention, same reaction conditions includes but not limited to: solution composition, constituent concentration, pH value, temperature, reaction times etc.
Advantage of the present invention: 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 coxsackie virus A 16-type of biotechnology field detects.
Description of drawings
Fig. 1 is nm gold particles and the product D NA hybridization synoptic diagram of coxsackie virus A 16-type after loop-mediated isothermal amplification that is marked with molecular probe, and nm gold particles is along coxsackie virus A 16-type product of loop-mediated isothermal amplification dna molecular chain formation aggregate.
Specific 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 coxsackie virus A 16-type particular sequence in one pipe, in the pipe be 250 μ l can increase in the detected sample coxsackie virus A 16-type DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the coxsackie virus A 16-type RNA in the detected sample that increases again after the reverse transcription process.
Contain the nm gold particles that diameter is 20 nm in the nm gold particles solution, concentration is 1.2 nM, 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, 0.4 M betaine (Sigma – Aldrich, St. Louis, MO), 10 mM MgSO4,1.4 mM dNTPs, 1 * ThermoPol reaction buffer (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'-CAGCTTACCATTGGGAAAAAAAAA-SH-3'。
The F3 primer sequence of coxsackie virus A 16-type:
5'-AAAGTCTCCAAGCGCCGA-3'。
The B3 primer sequence of coxsackie virus A 16-type:
5'-TGATACGTCAGGTCGAGTGG-3'。
The FIP primer sequence of coxsackie virus A 16-type:
5'-GCTTCCTGTGTCGTGATGGTGGGGTTACAGTGATCGTGTGGC-3'。
The BIP primer sequence of coxsackie virus A 16-type:
5'-ATAGCCTATGGGGAGTGGCCTGCTTGTCGACTGCCGTTG-3'。
Operating process is as follows: add nucleic acid or the biased sample that 10 μ l extract in the pipe that ring mediated isothermal nucleic acid amplification system is housed 1..
2. the pipe that mixture will be housed is put into the water-bath 30 minutes of 60 ° of C.
3. add nm gold particles solution in the pipe in step 2 again, this moment, mixed solution was red.
4. 60 ° of C water-baths are 15 minutes.
The color of solution in the pipe behind the observing response, solution keep the red constant coxsackie virus A 16-type that then do not contain to infect specific RNA or coxsackie virus A 16-type DNA; If solution becomes lilac, judge that then containing coxsackie virus A 16-type in the sample infects specific RNA or coxsackie virus A 16-type DNA.
SEQUENCE?LISTING
＜110〉Truehigh Tech Co., Ltd.
＜120〉a kind of test kit that detects coxsackie virus A 16-type
<130> Demo
<160> 5
<170> PatentIn?version?3.5
<210> 1
<211> 24
<212> DNA
<213> Human?coxsackievirus?A16
<400> 1
cagcttacca?ttgggaaaaa?aaaa 24
<210> 2
<211> 18
<212> DNA
<213> Human?coxsackievirus?A16
<400> 2
aaagtctcca?agcgccga 18
<210> 3
<211> 20
<212> DNA
<213> Human?coxsackievirus?A16
<400> 3
tgatacgtca?ggtcgagtgg 20
<210> 4
<211> 42
<212> DNA
<213> Human?coxsackievirus?A16
<400> 4
gcttcctgtg?tcgtgatggt?ggggttacag?tgatcgtgtg?gc 42
<210> 5
<211> 39
<212> DNA
<213> Human?coxsackievirus?A16
<400> 5
atagcctatg?gggagtggcc?tgcttgtcga?ctgccgttg 39
Claims (9)
1. one kind is detected the test kit of coxsackie virus A 16-type 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 coxsackie virus A 16-type particular sequence; (2) can increase in the detected sample coxsackie virus A 16-type DNA or earlier through the ring mediated isothermal nucleic acid amplification system of the coxsackie virus A 16-type 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 coxsackie virus A 16-type DNA in the loop-mediated isothermal amplification amplification detected sample or earlier through after the reverse transcription process with the coxsackie virus A 16-type 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 coxsackie virus A 16-type 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 coxsackie virus A 16-type.
4. according to claim 2, the amplification object of loop-mediated isothermal amplification can be the mixture of coxsackie virus A 16-type DNA, RNA or DNA and RNA in the detected sample.
5. according to claim 2, nm gold particles probe and coxsackie virus A 16-type 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 coxsackie virus A 16-type 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 coxsackie virus A 16-type product of loop-mediated isothermal amplification DNA hybridization cause solution or solid surface color change, this variation can directly be judged by naked eyes, also can utilize opticinstrument detections such as spectrophotometer.
8. according to claim 3, molecular probe is meant the functional molecular that can discern coxsackie virus A 16-type product of loop-mediated isothermal amplification DNA, and 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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011101557911A CN102226222A (en) | 2011-06-10 | 2011-06-10 | Kit for detecting coxsackie virus A16 |
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| CN2011101557911A CN102226222A (en) | 2011-06-10 | 2011-06-10 | Kit for detecting coxsackie virus A16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513310A (en) * | 2013-09-27 | 2015-04-15 | 中国科学院上海巴斯德研究所 | Preparation and application of anti-Coxsackie virus A16 monoclonal antibody |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659999A (en) * | 2009-08-04 | 2010-03-03 | 天津朝海科技有限公司 | Method for detecting product of loop-mediated isothermal amplification |
| CN101812544A (en) * | 2010-05-06 | 2010-08-25 | 天津朝海科技有限公司 | Influenza virus detection kit |
| CN101824492A (en) * | 2010-05-18 | 2010-09-08 | 天津朝海科技有限公司 | Kit for detecting AH1N1 influenza virus |
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2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659999A (en) * | 2009-08-04 | 2010-03-03 | 天津朝海科技有限公司 | Method for detecting product of loop-mediated isothermal amplification |
| CN101812544A (en) * | 2010-05-06 | 2010-08-25 | 天津朝海科技有限公司 | Influenza virus detection kit |
| CN101824492A (en) * | 2010-05-18 | 2010-09-08 | 天津朝海科技有限公司 | Kit for detecting AH1N1 influenza virus |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513310A (en) * | 2013-09-27 | 2015-04-15 | 中国科学院上海巴斯德研究所 | Preparation and application of anti-Coxsackie virus A16 monoclonal antibody |
| CN104513310B (en) * | 2013-09-27 | 2017-12-08 | 中国科学院上海巴斯德研究所 | A kind of preparation and its application of the monoclonal antibody of anti-coxsackie virus A 16-type |
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Application publication date: 20111026 |