CN105296675A - Nucleic acid detecting kit for influenza B viruses and preparation method of nucleic acid detecting kit - Google Patents
Nucleic acid detecting kit for influenza B viruses and preparation method of nucleic acid detecting kit Download PDFInfo
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- CN105296675A CN105296675A CN201510875348.XA CN201510875348A CN105296675A CN 105296675 A CN105296675 A CN 105296675A CN 201510875348 A CN201510875348 A CN 201510875348A CN 105296675 A CN105296675 A CN 105296675A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention belongs to the technical field of biology and particularly relates to a reverse transcription-recombinase polymerase amplification (RT-RPA) detecting kit for influenza B viruses and a preparation method of the detecting kit. The detecting kit is composed of an RT-RPA reaction system, an RNA enzyme inhibitor, an influenza B virus fragment NS positive plasmid Puc-NS, a negative quality control, a fragment NS RPA primer and an exo probe. A rapid, sensitive and specific isothermal real-time fluorescent nucleic acid detection method for influenza B viruses is established by using the specific primer and the probe and by preparing the RT-RPA reaction system and placing a Twista real-time fluorescent detection instrument for real-time fluorescent detection. The invention relates to application of the specific RPA primer and the exo probe to clinical differential diagnosis of influenza B virus infection and identification of a virus isolate strain.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Influenza B virus reverse transcription-recombinase polymerase amplification detection kit and preparation method thereof.
Background technology
Influenza B virus belongs to orthomyxoviridae family, and genome is made up of 8 minus strand single-stranded RNA sections, main infection people and sea dog.According to virus surface proteins haemagglutinin antigen sex differernce, Influenza B virus is divided into Victoria system and Yamagata system.Influenza B virus causes and breaks out with popular in crowd, it is mankind's seasonal current Influenza Virus Major Members, can all groups be infected, serious disease and even death can be caused to some special populations (as pregnant woman, the elderly and underlying disease patient).The detection method of Influenza B virus mainly comprises serology and detection of nucleic acids, and serological method mainly carries out antigens genotyping with standard serum to the virus strain be separated to, time and effort consuming, is not suitable for the diagnosis of clinical fast typing.Current nucleic acid detection method mainly comprises regular-PCR method and Real-timetimePCR method etc.Real-timetimePCR method has good specificity and susceptibility, but relies on expensive instrument and reagent; By contrast, regular-PCR susceptibility is poor and need agarose electrophoresis to carry out result judgement, is not suitable for the detection of extensive sample.Therefore, need to set up second type influenza virus nucleic acid detection method that is quick, responsive, that be convenient to popularization, to improve Influenza Surveillance and clinical diagnosis level.
Summary of the invention
The present invention needs the problem solved to be to provide a kind of Influenza B virus reverse transcription-recombinase polymerase amplification (RT-RPA) detection kit and preparation method thereof.
Influenza B virus RT-RPA detection kit provided by the invention is made up of RT-RPA reaction system, NS fragment positive plasmid Puc-NS, negative quality control product, NS fragment RPA primer and exo probe.
NS fragment RPA primer and exo probe are:
NS-F,5'-AAAGCCAATTCGAGCAGCTGAAACTGCGGTGG-3'；
NS-R,5`-CCATGTCAGCTATTATGGAGCTGTTAACTA-3`；
NS-exo-probe,5`-CCGATTATCACCAGAAGAGGGAGACAA[dT-FAM]TA[THF]AC[dT-BHQ]GGTCACGGAAGAAC[3`-block]-3`
The preparation method of Influenza B virus RT-RPA detection kit of the present invention:
(1) viral nucleic acid extracts: adopt phenol chloroform method to extract viral RNA, concrete steps: get clinical Nasopharyngeal swabs sample or viral cultures nucleic acid 100 μ L, add in the 1.5mlEP containing 200ul water-saturated phenol, concuss mixes, leave standstill 3min, add 200ul chloroform, fully shake mixing, centrifugal 15 minutes of 12000g room temperature; Aspirate supernatant, adds the Virahol of twice supernatant volume, then adds 1/10 supernatant volume 3M sodium-acetate 35-45ul, fully-20 DEG C of standing 2h after mixing; Take out at 4 DEG C, 12000g, centrifugal 20min; Abandon supernatant, add 75% ethanol 1ml, 4 DEG C, 12000g, centrifugal 20min; Abandon supernatant, drying at room temperature, add 40ulDEPC water dissolution RNA ,-70 DEG C of refrigerators are placed for subsequent use;
(2) RT-RPA reaction: the TwistAmpRTexos test kit adopting TwistDx company of Britain, get 29.5 μ L reaction buffers, add following component respectively: each 2.1 μ L of primer L-RPAF (10mM) and L-RPAR (10mM), 2.6 μ LRPAexo probes (10mM), 1 μ LRNA enzyme inhibitors (5U), 7.2 μ L ultrapure waters, 5 μ L sample RNA templates, vortex shakes, of short duration centrifugal, add 2.5 μ L magnesium acetates (280mM) again, mix centrifugal after, put into Twista real-time fluorescence detector, temperature of reaction is 40 DEG C, time is 20min.
Compared with prior art, the invention has the beneficial effects as follows:
Design is for L fragment RPA primer and exo probe, by reverse transcription-recombinase polymerase amplification method, temperature of reaction is under 40 DEG C of isothermal conditions, 20min can accompany thrombocytopenic syndromes virus sample and viral isolates to carry out nucleic acid molecule qualification to generating heat clinically, without the need to expensive plant and instrument, convenient operation, has the features such as quick, responsive, special, for heating companion's thrombocytopenic syndromes virus monitor and clinical economics provide a kind of new detection method.
Accompanying drawing explanation
Fig. 1 detects the screening of Influenza B virus NS sections RPA primer agarose gel electrophoresis
Embodiment
There is provided specific embodiment to set forth technical scheme of the present invention further below, but the application of the technology of the present invention is not limited to embodiment.
Embodiment 1: target gene plasmid construction
Design the Auele Specific Primer for Influenza B virus NS fragment, by PCR method amplification acquisition 806 gene fragment, reclaim being connected with carrier T after purifying, obtain target gene positive plasmid, called after Puc-NS.
Embodiment 2: primed probe design and screening
Analyzed by sequence analysis software, with Influenza B virus, comparatively NS fragment conservative region is for template, and design 9 forward primers and 10 reverse primers altogether, primer length is 30-33bp; Design 1 exo probe (called after NS-exo-probe), length is 48bp, wherein 28 bit base T mark fluorescent material FAM, and 31 bit bases are replaced by tetrahydrofuran (THF), 34 bit base mark fluorescent substance B HQ simultaneously, closes 3` terminal bases simultaneously.
Adopt TwistAmpBasickit (TwistDx company, Britain), with target gene cloned plasmids Puc-NS for template, take agarose gel electrophoresis as result indicatory device, the primer of above-mentioned design is screened.The evaluation index of primer quality comprises specificity, susceptibility, amplification efficiency and primer noise etc.Get 29.5 μ L reaction buffers, add following component respectively: each 2.4 μ L of upstream and downstream primer (10mM), 12.2 μ L ultrapure waters, 1 μ L plasmid Puc-L template.Add 2.5 μ L magnesium acetates (280mM) again.Mix centrifugal after, put into 40 DEG C, incubation 5min.Vortex concussion 8-10 time, of short duration centrifugal, put into 40 DEG C, reaction 20min.Get reaction product 2ul and put into 18ul water, with phenol-chloroform method, purifying is carried out to DNA.1.5% agarose gel electrophoresis detects purify DNA product, and result screens one group of primer, called after NS-F and NS-R (Fig. 1).
The susceptibility of embodiment 3:RT-RPA
Target gene cloned plasmids Puc-NS is diluted 100 times, then becomes 10 by 10 times of gradient dilutions
-2-10
-9totally eight concentration, as template, carry out RT-RPA reaction: adopt TwistAmpRTexos test kit (TwistDx company, Britain).Get 29.5 μ L reaction buffers, add following component respectively: each 2.1 μ L of primer NS-F (10mM) and NS-R (10mM), 2.6 μ LRPAexo probes (10mM), 1 μ LRNA enzyme inhibitors (5U), 7.2 μ L ultrapure waters, 5 μ L sample RNA templates.Vortex shakes, of short duration centrifugal.Add 2.5 μ L magnesium acetates (280mM) again.Mix centrifugal after, put into Twista real-time fluorescence detector, temperature of reaction is 40 DEG C, and the time is 20min.
The RT-RPA method set up in this experiment can Monitoring lower-cut be 100copies/ μ L, has good susceptibility.
The specificity of embodiment 4:RT-RPA
With influenza A virus, dengue fever virus, respiratory and enteric coronavirus, rhinovirus and respiratory syncytial virus nucleic acid for template, carry out RT-RPA experiment with the RPA primer designed and exo probe.Result does not all have fluorescent signal to detect to above-mentioned malicious nucleic acid, shows that the RT-RPA method set up has good specificity.。
Embodiment 5: clinical samples detects
Collect 65 parts of heating companion thrombocytopenic syndromes virus infection clinical samples, all samples all carry out virus purification by cell cultures.
(1) viral RNA extracts
Phenol chloroform method is adopted to extract viral RNA, concrete steps: 1. get Nasopharyngeal swabs sample 100 μ L, add in the 1.5mlEP containing 200ul water-saturated phenol, concuss mixes, and leaves standstill 3min, adds 200ul chloroform, abundant concussion mixing, centrifugal 15 minutes of 12000g room temperature.2. Aspirate supernatant, adds the Virahol of twice supernatant volume, then adds 1/10 supernatant volume 3M sodium-acetate and be about 40ul, fully-20 DEG C of standing 2h after mixing; Take out at 4 DEG C, 12000g, centrifugal 20min.3. abandon supernatant, add 75% ethanol 1ml, 4 DEG C, 12000g, centrifugal 20min.4. abandon supernatant, drying at room temperature, add 40ulDEPC water dissolution RNA.
(2) RT-RPA reaction: the TwistAmpRTexos test kit (TwistDx company, Britain) adopting company.Get 29.5 μ L reaction buffers, add following component respectively: each 2.1 μ L of primer NS-F (10mM) and NS-R (10mM), 2.6 μ LRPAexo probes (10mM), 1 μ LRNA enzyme inhibitors (5U), 7.2 μ L ultrapure waters, 5 μ L sample RNA templates.Vortex shakes, of short duration centrifugal.Add 2.5 μ L magnesium acetates (280mM) again.Mix centrifugal after, put into Twista real-time fluorescence detector, temperature of reaction is 40 DEG C, and the time is 20min.
65 parts of clinical samples as a result, RT-RPA detects positive has 43 parts, negative 22 parts, result and virus purification result completely the same.
Claims (2)
1. the Influenza B virus detection kit based on reverse transcription-recombinase polymerase amplification, it is characterized in that test kit is made up of RT-RPA reaction system, RNA enzyme inhibitors, M fragment positive plasmid Puc-NS, negative quality control product, NS fragment RPA primer and exo probe, NS fragment RPA primer and exo probe are:
NS-F,5'-AAAGCCAATTCGAGCAGCTGAAACTGCGGTGG-3'；
NS-R,5`-CCATGTCAGCTATTATGGAGCTGTTAACTA-3`；
NS-exo-probe,5`-CCGATTATCACCAGAAGAGGGAGACAA[dT-FAM]TA[THF]AC[dT-BHQ]GGTCACGGAAGAAC[3`-block]-3`。
2. the preparation method of a kind of Influenza B virus detection kit based on reverse transcription-recombinase polymerase amplification according to claim 1, is characterized in that being made up of following steps:
(1) viral nucleic acid extracts: adopt phenol chloroform method to extract viral RNA, concrete steps: get clinical Nasopharyngeal swabs sample or viral cultures nucleic acid 100 μ L, add in the 1.5mlEP containing 200ul water-saturated phenol, concuss mixes, leave standstill 3min, add 200ul chloroform, fully shake mixing, centrifugal 15 minutes of 12000g room temperature; Aspirate supernatant, adds the Virahol of twice supernatant volume, then adds 1/10 supernatant volume 3M sodium-acetate and be about 40ul, fully-20 DEG C of standing 2h after mixing; Take out at 4 DEG C, 12000g, centrifugal 20min; Abandon supernatant, add 75% ethanol 1ml, 4 DEG C, 12000g, centrifugal 20min; Abandon supernatant, drying at room temperature, add 40ulDEPC water dissolution RNA ,-70 DEG C of refrigerators are placed for subsequent use;
(2) RT-RPA reaction: adopt TwistAmpRTexos test kit, get 29.5 μ L reaction buffers, add following component respectively: each 2.1 μ L of primer L-RPAF10mM and L-RPAR10mM, 2.6 μ LRPAexo probe 10mM, 1 μ LRNA enzyme inhibitors 5U, 7.2 μ L ultrapure waters, 5 μ L sample RNA templates; Vortex shakes, of short duration centrifugal, then adds 2.5 μ L magnesium acetate 280mM; Mix centrifugal after, put into Twista real-time fluorescence detector, temperature of reaction is 40 DEG C, and the time is 20min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797245A (en) * | 2019-02-21 | 2019-05-24 | 中国人民解放军东部战区总医院 | It is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus |
| CN112195220A (en) * | 2020-10-13 | 2021-01-08 | 济南国益生物科技有限公司 | Lateral flow chromatography-recombinase constant-temperature amplification method for rapid detection of nucleic acid |
| CN113528707A (en) * | 2021-07-13 | 2021-10-22 | 国药(武汉)医学实验室有限公司 | Probe and kit for detecting influenza B virus and using method thereof |
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| CN102146485A (en) * | 2011-03-24 | 2011-08-10 | 武汉大学 | One-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for influenza virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102146485A (en) * | 2011-03-24 | 2011-08-10 | 武汉大学 | One-step RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit for influenza virus |
Non-Patent Citations (1)
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| 孙魁等: "重组酶聚合酶扩增技术的研究进展", 《军事医学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109797245A (en) * | 2019-02-21 | 2019-05-24 | 中国人民解放军东部战区总医院 | It is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus |
| CN112195220A (en) * | 2020-10-13 | 2021-01-08 | 济南国益生物科技有限公司 | Lateral flow chromatography-recombinase constant-temperature amplification method for rapid detection of nucleic acid |
| CN113528707A (en) * | 2021-07-13 | 2021-10-22 | 国药(武汉)医学实验室有限公司 | Probe and kit for detecting influenza B virus and using method thereof |
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Application publication date: 20160203 |