CN109797245A - It is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus - Google Patents
It is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus Download PDFInfo
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Abstract
The invention belongs to molecular Biological Detection fields, it is related to RPA primer, probe, kit and the application for detecting influenza B virus, the sequence of upstream and downstream primer is respectively B-F and B-R, probe sequence is as shown in SEQ ID NO.2, the RPA primer and probe of influenza B virus of the present invention can be used for quickly detecting influenza B virus and detect A type and influenza B virus simultaneously, and the downstream primer of the RPA amplification of influenza B virus of the present invention can be used as the PCR amplification primer of influenza B virus.Influenza B virus is detected using the present invention, time-consuming short, high sensitivity, high specificity, and can also quickly, accurate, specificity while detection A type and influenza B virus.
Description
Technical field
The present invention relates to technical field of molecular biological detection, and in particular to a kind of for detecting influenza B virus
RPA primer, probe, kit and application.
Background technique
Influenza is mainly the infectious respiratory disease as caused by influenza virus A type (FluA) and B-mode (Flu B), entirely
Ball death toll is up to 650,000.In general, influenza needs to carry out antiviral therapy in 48 hours after seizure of disease, and influenza is sick
The quick diagnosis of poison can substantially reduce the abuse of antibiotic, and implement necessary infection control measure.Under normal circumstances, doctor
Clinical symptoms of the meeting based on patient, realize the diagnosis of influenza virus.However, other viruses or bacterium can also cause influenza sample disease
Disease.Therefore, the diagnosis based on clinical symptoms easily causes the mistaken diagnosis of disease.Although virus purification culture is as diagnosis influenza virus
Gold standard, but 2-5 days turnaround time limited application of this method in clinical diagnosis and epidemiological survey.Quickly stream
The method that sense diagnostic test (RIDT) is most commonly used to diagnosis influenza virus.RIDT method has specificity high, and operation detects,
The TAT time is short.But the sensibility of RIDT is lower.Therefore, effectively accurately distinguish infection A type or influenza B virus for
Antiviral therapy is of great significance.
Since specific high and sensibility is good, molecular diagnostic techniques are widely used in the detection of influenza virus, especially exist
In clinical labororatory.Since viral RNA is denier in clinical sample.Therefore, it in the measurement of many molecular diagnosises, mentions
It taking, amplification and analysis are generally indispensable, during Molecular Detection, the specificity and spirit of the design of probe for detection
Sensitivity plays an important role.Polymerase chain reaction, that is, round pcr is a kind of common method of molecule amplification, but round pcr is to hard
Part equipment requirement is very high, and instrument is typically relatively expensive, and amplification rate is slow, therefore existing recombinase polymeric enzymatic amplification (RPA) skill
Art is come into being, and requirement of the RPA technology to hardware device is low, is a kind of isothermal amplification technique, is substantially increased reaction speed.
The key of RPA technology is the design of probe and primer, but probe used in PCR amplification and primer are not suitable for RPA technology mostly.
Summary of the invention
The present invention is intended to provide it is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus, this
The application at place had both included the method for the RPA primer of influenza B virus, probe for detecting influenza B virus, was also included
It is applied to while detecting influenza B virus and influenza A virus, further includes the RPA primer of influenza B virus in PCR
Application, provide it is quick, specific, delicately detect influenza B virus and detect A type and influenza B virus simultaneously
Method.
In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme:
One kind is for detecting influenza B virus RPA primer, the RPA primer nucleic acid sequence are as follows: upstream primer: B-F:
5 '-TCTGCTGGAATTGAAGGGTTTGAGCCATAC-3 ', downstream primer: B-R:5 '-
TCAAACGGAACTTCCCTTCTTTCTGAGTGT-3'；
For one kind for detecting influenza B virus RPA probe, the nucleic acid sequence of the RPA probe is SEQ ID NO.2:
5’-GTTCCTCAAATAGCAACTGTCCGAAATACAAT-THF-GGACCGATTACCCTT-3’。
It is a kind of for detecting the multiple RPA primer of influenza B virus and influenza A virus, including influenza B simultaneously
The primer and probe of viral RPA further includes the RPA primer and probe of influenza A virus.
Preferably, the RPA primer sequence of the influenza A virus are as follows: upstream primer sequence are as follows: MF146:5 '-
GGCTCTCATGGAATGGCTAAAGACAAGAC-3 ', downstream primer sequence are as follows: MR425:5 '-
TTGTATATGAGGCCCATGCAACTGGCAAGTG-3 ', the probe sequence of the influenza A virus are as follows: SEQ ID
NO.1:5 '-TTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTG-3 '.
Containing for detect influenza B virus RPA primer or probe or containing for and meanwhile detect influenza B virus with
The kit or Test paper of the multiple RPA primer and probe of influenza A virus.
A method of detection influenza B virus, comprising the following steps: (1) extract the RNA of virus to be measured；(2) it synthesizes
For detecting the probe of influenza B virus, using the RNA for extracting sample to be tested as template, reverse transcription reaction is carried out, synthesizes cDNA,
It is then expanded using influenza B virus RPA amplification system, RPA amplification is using primer and the spy for detecting influenza B virus
Needle；(3) above-mentioned amplified production is analyzed.
Method that is a kind of while detecting A type and influenza B virus, comprising the following steps: (1) extract virus to be measured
RNA；(2) RPA is expanded: the multiple RPA primer and probe for detecting influenza A virus and influenza B virus is synthesized, to mention
The RNA of the sample to be tested taken is template, carries out reverse transcription reaction, then using A type and the dual RPA amplification of influenza B virus
System amplification, dual RPA amplification is using for detecting the primer and probe of influenza A virus and for detecting influenza B
The modifier of the primer and probe of virus, the A type and influenza B virus probe is different；(3) above-mentioned amplification is analyzed to produce
Object.3 ' ends of the probe of the A type and influenza B virus are marked with an arm Spacer (C3), influenza A virus respectively
The thymidine of 33 positions of 32 positions and influenza B virus probe sequence of probe sequence replaces with tetrahydrofuran.
Preferably, it while detecting in A type and the method for influenza B virus step (3) analysis method and uses nucleic acid immunization
Sidestream chromatography test paper is tested and analyzed.Nucleic acid immunization Sidestream chromatography method does not have to using excessively complicated instrument, and operates letter
Just, visual result.
Preferably, under the RPA of method that is above-mentioned while detecting A type and influenza B virus, A type and influenza B virus
5 ' the ends label of trip primer has, i.e. Biotin, the modification of the RPA probe for detecting A type and influenza B virus
Object is respectively fluorescein FITC and digoxin DIG, is sprayed respectively in two detection lines on nucleic acid immunization Sidestream chromatography test paper glimmering
Light element antibody A nti-FITC and DigiTAb Anti-DIG are coated with ox in gold-labelled pad on nucleic acid immunization Sidestream chromatography test paper
Haemocyanin BSA, sprayed biological element bovine serum albumin(BSA) on nature controlling line, i.e. B-BSA.
Preferably, method that is above-mentioned while detecting A type and influenza B virus, the concentration of Anti-FITC are to be at least
The concentration of 12.5ng/strip, Anti-DIG are at least 75ng/strip, and the concentration of Biotin-BSA is at least 50ng/strip；
The nucleic acid Sidestream chromatography time is at least 5 minutes；The temperature of dual RPA amplification is 35-45 DEG C；The time of dual RPA amplification is at least
It is 5 minutes；It is each that the primer concentration used of dual RPA amplification is respectively as follows: influenza A virus RPA upstream and downstream primer concentration are as follows:
50-200nM, influenza B virus RPA upstream and downstream primer concentration are each are as follows: 50-200nM.Multiple RPA system parameter and nucleic acid effluent
Chromatography parameter maintains the purpose that can reach detection in the range.
Preferably, method that is above-mentioned while detecting A type and influenza B virus, wherein the concentration of Anti-FITC be
The concentration of 50ng/strip, Anti-DIG are 250ng/strip, and the concentration of Biotin-BSA is 50ng/strip nucleic acid effluent layer
The analysis time is at least 20 minutes；The temperature of dual RPA amplification is 39 DEG C；The time of dual RPA amplification is 20 minutes；Dual RPA
It is each that the primer concentration used of amplification is respectively as follows: influenza A virus RPA upstream and downstream primer concentration are as follows: 100nM, influenza B disease
Malicious RPA upstream and downstream primer concentration is each are as follows: 150nM.Multiple RPA system parameter and nucleic acid Sidestream chromatography parameter maintain the value can be
Clearly testing result is obtained in short period.
Preferably, the RPA downstream primer B-R for detecting influenza B virus of the invention is in addition to can be used as RPA amplification
Primer outside, be also used as the downstream primer of PCR.
Beneficial effects of the present invention are as follows: the present invention provides one kind for detecting influenza B virus RPA primer and spy
Needle can carry out quick RPA amplification using the reverse transcription cDNA of the primer and probe to influenza B virus, meanwhile, it can also should
The probe and primer combination for inventing discovery are applied on kit or Test paper for fast and easily inspection influenza B disease
Poison.A type and B-mode is detected the present invention also provides the method for individually detecting influenza B virus with the primer and probe and simultaneously
The sensitivity of the method for influenza virus, the method detection of the invention is very high, and high specificity is time-consuming short, and because being that the RPA used expands
Increase and nucleic acid Sidestream chromatography technology, do not need it is frequent as PCR must heat up, therefore do not need special equipment, operate also compared with
For simplicity.
Detailed description of the invention
Fig. 1 RPA expands schematic diagram and nucleic acid Sidestream chromatography test paper detection figure
Influence experimental result picture of Fig. 2 antibody concentration to nucleic acid immunization Sidestream chromatography result
Fig. 3 chromatographs the time to the influence experimental result picture of nucleic acid immunization Sidestream chromatography result
Fig. 4 detects influenza B virus and influenza A virus system optimization experimental result picture simultaneously
Fig. 5 detects specificity experiments result figure
Fig. 6 detection sensitivity experimental result picture
Specific embodiment
According to non-structural protein (the Flu B NS) gene for the influenza B virus delivered in GeneBank database, benefit
Sequence alignment is carried out with software DNAMAN, design primer is as follows with probe:
Wherein, B-F is upstream primer, and B-R is downstream primer, and SEQ ID NO.2 is probe.
The label of the RPA probe of influenza B virus can be fluorophor or drug or photosensitizer or medicine contrast agent.
In the present embodiment, choosing digoxin (DIG) is its modifier.
In the present embodiment the 5 ' ends and 3 ' ends of the RPA probe of influenza B virus used mark respectively digoxin (DIG) and
Between arm Spacer (C3), 33 position thymidine of sequence replaces with tetrahydrofuran, i.e., are as follows: DIG-GTTCCTCAAATAGCAACTG
TCCGAAATACAAT-THF-GGACCGATTACCCTT-spacer(C3)。
The RPA downstream primer B-R of influenza B virus used is marked with biotin, as Biotin- in the present embodiment
TCAAACGGAACTTCCCTTCTTTCTGAGTGT-3'.So that the product after amplification has double labelling.
Mark fluorescent is plain (FITC) respectively at the 5 ' ends and 3 ' ends of the RPA probe of influenza A virus used in the present embodiment
With an arm Spacer (C3), 32 position thymidine of sequence replaces with tetrahydrofuran, as FITC-
TTCACGCTCACCGTGCCCAGTGAGCGAGGAC-THF-GCAGCGTAGACGCTTTG-spacer(C3)。
The RPA downstream primer MF425 of influenza A virus used is marked with biotin, as Biotin- in the present embodiment
TTGTATATGAGGCCCATGCAACTGGCAAGTG-3’。
The RPA downstream primer B-R of the influenza B virus is also used as PCR other than it can be used as the primer of RPA amplification
Downstream primer.
The RPA probe and primer of influenza B virus of the present invention can be used for kit or Test paper, are used to quick
Detect influenza B virus.It is of course also possible to by the RPA probe of other viruses and primer and influenza B virus of the invention
Probe and primer combination, such as the RPA probe and primer of influenza A virus, for distinguishing other viruses and influenza B disease
Poison.
The method for detecting influenza B virus and influenza A virus simultaneously
Influenza B virus and influenza A virus are detected with the RPA probe of influenza B virus of the invention and primer
Method, comprising the following steps: 1, prepare nucleic acid Sidestream chromatography pad；2, viral RNA is extracted；3, to carrying out RPA after RNA reverse transcription
Amplification, RPA amplification probe and primer are above-mentioned influenza B virus probe and influenza A probe；4, amplified production is taken,
Buffer is added, is added dropwise in nucleic acid immunization Sidestream chromatography test paper, visual results.
All solvents and reagent that the present embodiment uses are commercially available finished product, and primer and probe are the raw work biotechnology in Shanghai
Co., Ltd (Shanghai) synthesis；Virus RNA extraction kit is purchased from Tiangeng biochemical technology Co., Ltd (Beijing)；TwistAmp
Nfo Kit is purchased from TwistDx company (Britain)；RPA dry powder is purchased from TwistDx company (Britain)；PCR amplification reagent, reverse transcription
Enzyme A MV Reverse Transcriptase XL and RNase inhibitor is purchased from Dalian treasured biotech firm.
The step of preparing Sidestream chromatography test paper:
(1) preparation and assembling of nucleic acid Sidestream chromatography test paper
For the nucleic acid immunization Sidestream chromatography immune test paper of double-stranded DNA detection, sample is pasted in plastic backings in order
Pad, colloidal gold composite pad, cellulose acetate film and blotting paper, the overlapped 2mm of each section.
The colloid gold particle of 40nm is prepared using reduction of sodium citrate gold chloride, and it is affine to carry out strepto- to colloid gold particle
Element modification, the specific steps are as follows:
1. sequentially adding 99mL ultrapure water and 1mL gold chloride (1%) in clean glass triangle flask, it is heated to boiling
Afterwards, 1mL trisodium citrate (1%) is added, lasting to stir, solution becomes red, and remains unchanged, continue heating stirring 15min,
It is cooled to room temperature, it is spare；
2. taking 1ml colloidal gold, 1 μ L 200mM dobell's solution is added, adjusts pH to 9.5；
3. taking 5 μ L strepto- nucleophilic nucleins (1mg/ml), it is added in 495 μ L dobell's solutions (2mM), is uniformly mixed；
4. the strepto- nucleophilic nuclein solution after dilution is added dropwise in colloidal gold solution, mix, after being all added, by it
It is placed on vertical rotary blending instrument, mixes 2h；
5. taking 80 μ L concentration is that 20% bovine serum albumin solution (being dissolved in the dobell's solution of 2mM) is added to
It states in colloidal gold solution, continues to mix 30min；
6. above-mentioned solution 2000rpm is centrifuged 10min, supernatant is shifted into new centrifuge tube, 12000rpm centrifugation
10min removes supernatant, and 200 μ L re-suspension liquids are added and (15mM Tris pH 9.5, include sucrose 2%-5%, PEG2000
0.5%-2%, BSA0.5%, tween-200.05%), concussion mixes, and is sprayed in gold-labelled pad, and ambient temperature overnight is dry.
7. by anti-fluorescein antibody (Anti-FITC), DigiTAb (Anti-DIG) and biotinylation BSA (B-BSA), benefit
It is diluted with antibody diluent (100mM sodium bicarbonate, 5% methanol, 2% sucrose), then the velocity spray of 2 μ L/cm is in acetic acid fibre
It ties up on plain film, 37 DEG C of dry 1h.
A figure is dual RPA amplification schematic diagram in attached drawing 1, and FluA is influenza A virus；Flu B is influenza B virus；
By the primer and probe of design synthesis, the reverse transcription nucleic acid sequence of the two is expanded.B figure is nucleic acid immunization Sidestream chromatography
Schematic diagram, sample drop is added in sample pad, followed by gold-labelled pad, two detection lines and nature controlling line.FITC is fluorescence in figure
Element label, DIG are digoxigenin labeled, and Nfo is endonuclease IV restriction endonuclease, and Biotin is biotin labeling, THF tetra-
Hydrogen furans replaces, and SA-AU is Streptavidin gold, and Biotin-BSA is biotinylation bovine serum albumin(BSA), and Anti-DIG is ground
Digoxin antibody, Anti-FITC are anti-fluorescein antibody.The strepto- on biotin labeling and gold-labelled pad that amplified production is had is affine
Plain gold continues flow forward after combining, and when flowing through detection line, amplified production conjugate institute is in hapten-marked and detection line
Antibody, which combines, to be shown, and when finally flowing through nature controlling line, amplified production conjugate shows in conjunction with the B-BSA on nature controlling line.
Nucleic acid Sidestream chromatography test paper antibody concentration and the time-optimized experiment of chromatography
Performance evaluation is carried out to this nucleic acid Sidestream chromatography test paper, chooses the antibody concentration of best chromatography time and spraying.It is excellent
Change method is the double-stranded DNA that synthesis is respectively provided with haptens (FITC or DIG) and biotin labeling, is used after being diluted to various concentration
The test paper tests it, and different test paper antibody concentrations are as follows:
The different test paper antibody concentration table of table 1
| Test paper serial number | B-BSA(ng/strip) | Anti-DIG(ng/strip) | Anti-FITC(ng/strip) |
| 1 | 50 | 75 | 12.5 |
| 2 | 100 | 150 | 25 |
| 3 | 200 | 250 | 50 |
Experiment is divided into three groups:
Negative control group (Negative control): the double-stranded DNA of biotin labeling is only had；
DIG detection group (DIG-labeled DNA): the double-stranded DNA with biotin labeling and haptens DIG；
FITC detection group (FITC-labeled DNA): the double-stranded DNA with biotin labeling and haptens FITC；
Three groups of DNA are dripped respectively on three parts of chromatography detecting test papers, observation as a result, experimental result such as Fig. 2, from left to right according to
Secondary is negative control group, DIG detection combination FITC detection group, title of the test paper left side by applying antibody on test paper, the number on the right
For the concentration of antibody or biotinylation bovine serum albumin(BSA).Result of study shows to reach in above-mentioned three groups of antibody concentrations
To testing goal, but optimum antibody label concentration is respectively B-BSA:50ng/strip, Anti-DIG:250ng/strip,
Anti-FITC:50ng/strip.
In order to reach better testing result, the present invention is also optimized the time of nucleic acid Sidestream chromatography.Specific core
The sour time-optimized experiment embodiment of Sidestream chromatography are as follows: synthesis is respectively provided with haptens (FITC or DIG) and biotin labeling
Double-stranded DNA, be equally divided into three groups of experiments: negative control group, DIG detection group and FITC detection group drip three groups of DNA respectively three
On part Sidestream chromatography Test paper, tomographic results of each group of chromatography assay at 5 minutes, 20 minutes, 35 minutes are recorded, are tied
Fruit such as Fig. 3.From the results, it was seen that at 5 minutes, it is already possible to judge tomographic results, when by 20 minutes, as a result
Through clearly, very big difference is had no when result when 35 minutes was compared with 20 minutes, therefore chromatograph the time to can be controlled in 5
Minute or more, 20 minutes are best.
The RT-RPA amplification system of the method and step 3 of influenza B virus and influenza A virus is detected simultaneously
RT-RPA amplification system (25 μ L): taking the 29.5 μ l of buffer in TwistAmp nfo Kit, and one pipe RPA of dissolution is dry
Powder is then distributed into two pipes, every 14.75 μ L of pipe.A type and B-mode RPA primer and probe are sequentially added in 0.2mL centrifuge tube,
The aforementioned 14.75 μ L of buffer, template cDNA is 2 μ L, and after mixing, MgAc (280mM) 1.25 μ L is added.Centrifuge tube is set
It after being incubated for a period of time under certain temperature, mixes, a period of time is then reacted under certain temperature.Take 5 μ L that 70 μ L PBST are added
In, using nucleic acid immunization Sidestream chromatography test paper, (antibody concentration is followed successively by B-BSA:50ng/strip, Anti-DIG:250ng/
Strip, Anti-FITC:50ng/strip) it is analyzed, testing result is recorded after 15min.
RT-RPA amplification system reaction temperature, reaction time, the experiment of primer and probe concentration optimization
The final concentration of 100nM of the influenza A primers being added in above-mentioned amplification system, influenza A probe SEQ
The final concentration of 40nM of ID NO.1, the final concentration of 100nM of influenza B virus primer, influenza B virus probe are final concentration of
40nM.Different incubation temperatures is set, successively are as follows: 20,35,37,39,41,45,50 DEG C, reaction time 20min.5 μ L are taken to expand
Increase production object, 70PBST solution is added, nucleic acid Sidestream chromatography test paper measures 15min, records result such as Fig. 4 A.
In above-mentioned amplification system, influenza A primers, the 40nM Flu-A disease of final concentration of 100nM are sequentially added
Malicious probe SEQ ID NO.1,100nM influenza B virus primer and 40nM probe, reaction temperature are 39 DEG C, and reaction is successively are as follows:
5,10,15,20,25min take 5 μ L amplified productions, and 70PBST solution is added, and nucleic acid Sidestream chromatography test paper measures 15min, record
As a result such as Fig. 4 B.
The primer and probe of different final concentrations is sequentially added in above-mentioned RT-RPA amplification system, as shown in table 2,39 DEG C incubate
20min is educated, 5 μ L amplified productions are taken, 70PBST solution is added, nucleic acid Sidestream chromatography test paper measures 15min, and record result is as schemed
4C。
The different probe of table 2 and primer final concentration
MF146 and MR425 is respectively influenza A virus RPA upstream and downstream primer in table, and SEQ ID NO.1 is Flu-A
Viral RPA probe, B-F and B-R are respectively influenza B virus RPA upstream and downstream primer, and SEQ ID NO.2 is influenza B virus
RPA probe.A1-A4 group and the experiment of B1-B4 group are respectively provided with to detect, A1-A4 group is to detect the primer of influenza A virus
Optimal final concentration, B1-B4 group experiment for detection influenza B virus primer optimal final concentration.
A figure is RPA system temperature optimization lab diagram in Fig. 4, and B figure is the time-optimized lab diagram of RPA system, and C figure is RPA body
It is concentration optimization lab diagram.For analysis chart 4 it is found that the temperature of amplification system is between 35-45 DEG C, 39 DEG C of temperature are best；Expand
Increase the time at 5 minutes or more, amplification can be perfectly clear to obtain testing result for 20 minutes；Each virus upstream and downstream primer is dense eventually
Degree is in 50-200nM, it is still further preferred that A type stream influenza A virus RPA upstream and downstream primer final concentration is each are as follows: 100nM, second
Type influenza virus RPA upstream and downstream primer final concentration is each are as follows: 150nM.
The specificity analysis experiment of detection
Following virus, first are expanded using the Optimal system for the RPA amplification for detecting A type and influenza B virus simultaneously respectively
Type influenza virus H1N1, influenza A virus H3N2, influenza B virus, Respiratory Syncytial Virus(RSV) (A type and Type B), people's tuberculosis
Malicious (MPV), human corona virus (CoV) and negative control (water) are then detected using nucleic acid immunization Sidestream chromatography test paper special
Property, antibody concentration, detection time are all made of Optimal system on nucleic acid immunization Sidestream chromatography test paper.Experimental result as shown in figure 5, from
It is left-to-right successively are as follows: negative control group (Negative control)；Influenza virus H1N1 detection group (Flu A H1N1),
Influenza A virus H3N2 detection group (Flu A H3N2), influenza B virus detection group (Flu B), A type subtype influenza virus
With influenza B virus (Flu A H1N1+Flu B), A type subtype influenza virus and influenza B virus (Flu A H3N2+
Flu B, Respiratory Syncytial Virus(RSV) A type (RSV A), Respiratory Syncytial Virus(RSV) Type B (RSV B), people's Pneumovirinae (MPV), people are coronal
Viral (CoV).The result shows that dual RPA successfully detects H1N1, H3N2 and Flu B, to RSVA, RSVB, MPV and CoV without anti-
Ying Xing.
The sensitivity analysis of detection is tested
The viral RNA of sufficient amount is prepared first, and preparation method includes four steps: (1) extracting viral nucleic acid；(2)cDNA
Synthesis；(3) PCR amplification；(4) synthesis of single stranded RNA.Concrete operations mode is as follows:
(1) virus is extracted
Reference virus RNA extracts kit (Tiangeng is biochemical, Code No.DP315-R) specification, the side chromatographed using column
Method extracts the RNA in oropharyngeal swab specimen, specific as follows: taking 1.0mL oropharyngeal swab specimen, 12000r/min is centrifuged 20min, removal
After 860 μ L supernatants, 560 μ L Carrier RNA working solutions are added, be vortexed concussion 15s, is then placed at room temperature for 15min, of short duration
After centrifugation, 560 μ L dehydrated alcohols are added, shakes 15s, is transferred in adsorption column (RNase-Free adsorption column CR2), 8000rpm
1min, abandon waste liquid, adsorption column is put back in collecting pipe, solution GD and RW is separately added into and is cleaned, be eventually adding 60 μ L without
RNase water elution.
(2) cDNA is synthesized
Reverse transcription (RT) reaction system (20 μ L): in the PCR pipe of 0.2 μ L, 5 μ L RNA, 1.0 μ L are separately added into
Random Primer(0.5μg/μL)、Reverse Transcription 10×Buffer 2μL、dNTP Mixture
(10mM) 2 μ L, 0.25 μ L of AMV Reverse Transcriptase XL, 0.25 μ L of RNase Inhibitor, supply nothing
RNase water is to 20 μ L.Reaction condition: 20 DEG C of 10min, 42 DEG C of 30min.
(3) PCR amplification
PCR amplification system (25 μ L): 2 × PCR Mix, 12.5 μ L, upstream primer (10 μM) and downstream primer (10 μM) are each
0.5 μ L, cDNA2 μ L, supplies no RNase water to 25 μ L.Primer pair AMPB/AMPC and B-F2/B-R is respectively adopted and carries out PCR expansion
Increase.Reaction condition: 95 DEG C of 5min；95℃ 30s,50℃ 30s,72℃ 30s；95℃ 30s,55℃ 30s,72℃ 30s；
72℃ 10min.PCR product is subjected to 1.2% agarose gel electrophoresis analysis, gel extraction, and is sequenced.
(4) synthesis of single stranded RNA
In the PCR pipe of 0.2mL, 10 × Transcription Buffer 2 μ L, ATP (50mM) 2 μ L is sequentially added,
GTP (50mM) 2 μ L, CTP (50mM) 2 μ L, UTP (50mM) 2 μ L, RNase Inhibitor (40U/ μ L) 0.5 μ L,
2 μ L of T7RNAPolymerase (50U/ μ L), 5 μ L purified pcr products, 42 DEG C of reaction 2h.After room temperature is cooling, RNase is added
Free DNase I (5U/ μ L) 2 μ L, 37 DEG C of 2h.Finally, purifying obtains single stranded RNA, RNA is measured using micro-spectrophotometer
Concentration.
Then distinguish the influenza A virus of gradient dilution synthesis and the single stranded RNA of influenza B virus, concentration is followed successively by 1
×107、1×106、1×105、1×104、1×103、1×102、1×101Copy/μ L takes 5 μ L to carry out cDNA synthesis respectively, and
After take 2 μ L cDNA to carry out dual RPA amplification and the detection of Sidestream chromatography test paper, and in triplicate.In addition, by A type and B-mode stream
Influenza Virus single stranded RNA isoconcentration mixing, then carry out cDNA synthesis, dual RPA amplification, RPA amplification system be 25 μ L, respectively plus
Enter the influenza A virus upstream and downstream primer and probe of final concentration difference 100nM and 40nM, the influenza B disease of 150nM and 40nM
Malicious upstream and downstream primer and probe.Reaction time is 39 DEG C of 20min.5 μ L RPA amplified productions are taken, it is mixed that 70 μ LPBS-T solution are added
After conjunction, sensitivity technique analysis is carried out with nucleic acid immunization Sidestream chromatography test paper, nucleic acid immunization Sidestream chromatography test paper detection temperature is
Room temperature, the concentration of the upper Anti-FITC sprayed is 50ng/strip on test paper, and the concentration of Anti-DIG is 250ng/strip, B-
The concentration of BSA is 50ng/strip, and the nucleic acid Sidestream chromatography time is 20 minutes.As a result such as Fig. 6, A figure is independent detection A type stream
Influenza Virus sensitivity experiments result figure, B figure are independent detection influenza B virus sensitivity experiments result figure, and C figure is while examining
Survey A type and influenza B virus sensitivity experiments figure.The lowest detection of A type and influenza B virus is individually detected as the result is shown
Limit is 50 copies/reaction；The detection limit for detecting A type and influenza B virus simultaneously is respectively 500 and 50 copies/react.
SEQUENCE LISTING
<110>Chinese People's Liberation Army's eastern theater of war hospital general
<120>a kind of for detecting RPA primer, probe, kit and the application of influenza B virus
<130> 2019.01.30
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213>artificial synthesized
<400> 1
tctgctggaa ttgaagggtt tgagccatac 30
<210> 2
<211> 30
<212> DNA
<213>artificial synthesized
<400> 2
tcaaacggaa cttcccttct ttctgagtgt 30
<210> 3
<211> 48
<212> DNA
<213>artificial synthesized
<400> 3
gttcctcaaa tagcaactgt ccgaaataca attggaccga ttaccctt 48
<210> 4
<211> 29
<212> DNA
<213>artificial synthesized
<400> 4
ggctctcatg gaatggctaa agacaagac 29
<210> 5
<211> 31
<212> DNA
<213>artificial synthesized
<400> 5
ttgtatatga ggcccatgca actggcaagt g 31
<210> 6
<211> 49
<212> DNA
<213>artificial synthesized
<400> 6
ttcacgctca ccgtgcccag tgagcgagga ctgcagcgta gacgctttg 49
Claims (12)
1. a kind of for detecting the RPA primer of influenza B virus, which is characterized in that the RPA primer nucleic acid sequence are as follows: upstream
Primer: B-F:5 '-TCTGCTGGAATTGAAGGGTTTGAGCCATAC-3 ', downstream primer: B-R:5 '-
TCAAACGGAACTTCCCTTCTTTCTGAGTGT-3’。
2. one kind is for detecting influenza B virus RPA probe, which is characterized in that the nucleic acid sequence of the RPA probe is SEQ
ID NO.2:5 '-GTTCCTCAAATAGCAACTGTCCGAAATACAATTGGACCGATTACCCTT-3 '.
3. a kind of for detecting the multiple RPA primer and probe of influenza B virus and influenza A virus simultaneously, feature exists
In, including primer described in claim 1 and probe as claimed in claim 2, further include influenza A virus RPA primer and
Probe.
4. according to claim 3 a kind of for detecting the multiple RPA of influenza B virus and influenza A virus simultaneously
Primer and probe, which is characterized in that the RPA primer nucleic acid sequence of the influenza A virus are as follows: upstream primer sequence are as follows:
MF146:5 '-GGCTCTCATGGAATGGCTAAAGACAAGAC-3 ', downstream primer sequence are as follows: MR425:5 '-TTG
TATATGAGGCCCATGCAACTGGCAAGTG-3 ', the probe sequence of the influenza A virus are as follows: SEQ ID NO.1:
5’-TTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCTTTG-3’。
5. the kit containing primer described in any one of claim 1-4 claim or probe.
6. a kind of method for detecting influenza B virus, which comprises the following steps:
(1) RNA of virus to be measured is extracted；
(2) RPA is expanded: synthesis claim 1 and primer and probe as claimed in claim 2 are to extract the RNA of sample to be tested
Template carries out reverse transcription reaction, synthesizes cDNA, is then expanded using influenza B virus RPA amplification system, and RPA amplification uses
Primer described in claim 1 and probe as claimed in claim 2；
(3) above-mentioned amplified production is analyzed.
7. a kind of method for detecting A type and influenza B virus simultaneously, which comprises the following steps:
(1) RNA of virus to be measured is extracted；
(2) RPA is expanded: synthesis is as claimed in claim 4 for detecting the multiple RPA of influenza A virus and influenza B virus
Primer and probe carries out reverse transcription reaction using the RNA of the sample to be tested of extraction as template, then uses A type and influenza B
Virus dual RPA amplification system amplification, dual RPA amplification use primer and probe as claimed in claim 4, the A type and
The modifier of influenza B virus probe is different, between 3 ' ends of the probe of the A type and influenza B virus are marked with respectively
Arm Spacer (C3), the chest of 33 positions of 32 positions and influenza B virus probe sequence of influenza A probe sequence
Gland pyrimidine replaces with tetrahydrofuran；
(3) above-mentioned amplified production is analyzed.
8. method that is according to claim 7 while detecting A type and influenza B virus, which is characterized in that step (3)
Analysis method is tested and analyzed using nucleic acid immunization Sidestream chromatography test paper.
9. method that is according to claim 8 while detecting A type and influenza B virus, which is characterized in that for detecting
5 ' the ends label of A type and the RPA downstream primer of influenza B virus has, for detecting A type and influenza B disease
The modifier of the RPA probe of poison is respectively FITC and DIG, is sprayed respectively in two detection lines on nucleic acid immunization Sidestream chromatography test paper
Anti-FITC and Anti-DIG is applied, BSA is coated in gold-labelled pad on nucleic acid immunization Sidestream chromatography test paper, sprays B- on nature controlling line
BSA。
10. method that is according to claim 9 while detecting A type and influenza B virus, which is characterized in that nucleic acid is exempted from
The concentration of the Anti-FITC sprayed on epidemic disease Sidestream chromatography test paper is to be at least 12.5ng/strip, and the concentration of Anti-DIG is at least
For 75ng/strip, the concentration of B-BSA is at least 50ng/strip, and the nucleic acid Sidestream chromatography time is at least 5 minutes；Dual RPA
The temperature of amplification is 35-45 DEG C, and the time of dual RPA amplification is at least 5 minutes, the primer concentration used of dual RPA amplification
Being respectively as follows: influenza A virus RPA upstream and downstream primer final concentration is respectively 50-200nM, influenza B virus RPA upstream and downstream primer
Final concentration is respectively 50-200nM.
11. method that is according to claim 9 while detecting A type and influenza B virus, which is characterized in that nucleic acid is exempted from
The concentration of the Anti-FITC sprayed on epidemic disease Sidestream chromatography test paper is 50ng/strip, and the concentration of Anti-DIG is 250ng/
The concentration of strip, B-BSA are 50ng/strip, and the nucleic acid Sidestream chromatography time is 20 minutes；The temperature of dual RPA amplification is 39
DEG C, the time of dual RPA amplification is 20 minutes, and the primer concentration used of dual RPA amplification is respectively as follows: influenza A virus
RPA upstream and downstream primer final concentration is respectively 100nM, and influenza B virus RPA upstream and downstream primer final concentration is respectively 150nM.
12. a kind of for detecting the application of influenza B virus RPA primer, which is characterized in that the downstream primer B-R of the primer:
5 '-TCAAACGGAACTTCCCTTCTTTCTGAGTGT-3 ' are alternatively arranged as the PCR primer of influenza B virus.
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