CN107385110A - A kind of RPA primers and its detection method for being used to detect the type of aviadenovirus serum 4 - Google Patents
A kind of RPA primers and its detection method for being used to detect the type of aviadenovirus serum 4 Download PDFInfo
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Abstract
The invention discloses a kind of RPA primers for being used to detect the type of aviadenovirus serum 4, the nucleotide sequence of the primer is as shown in SEQ ID NO.1 and SEQ ID NO.2.The invention also discloses a kind of RPA methods for being used to detect the type of aviadenovirus serum 4, this method mainly includes：The steps such as primer synthesis, testing sample DNA extractions, RPA amplified reactions and the analysis of RPA amplified productions.The RPA primer specificities of the present invention are strong, high sensitivity, and testing result is accurate.The detection method of the present invention is simple to operate, and stability is good, provides a kind of cost low, quick, special field diagnostic method with identification chicken gizzard inflammation hydropericardium syndrome for effective detection.
Description
Technical field
The invention belongs to molecular Biological Detection field, and in particular to for detecting the RPA primers of the type of aviadenovirus serum 4
And its detection method.
Background technology
Chicken gizzard inflammation caused by the type of aviadenovirus serum 4 (Fowl adenovirus serotype4, FAdV-4)-pericardium product
Liquid syndrome is highly contagious disease, and the disease takes place mostly in 3-10 week old broiler chicken, and 4-5 week old is the peak mortality phase, dead
Rate reaches as high as 80%, and very big economic loss is brought for China's aviculture.Quick field diagnostic, Neng Gouwei are carried out to this disease
The quality time is striven in effective prevention and control of epidemic disease, reduces the risk of loss and epidemic disease diffusion to greatest extent.
The method of detection aviadenovirus mainly has Virus Isolation, neutralization test, electron microscope method, agar to expand at present
Dissipate experiment, ELISA, PCR etc..Virus Isolation and virus neutralization tests need specific cell culture, and time-consuming；Electronic display
Micro mirror needs expensive instrument and not can determine that virus serotype；The sensitiveness of agar gel diffusion test is not high enough；ELISA is generally used
It is complex for operation step in the antibody response for determining specific adenovirus, while it is also required to specific instrument；Conventional polymerase chain is anti-
(Polymerase Chain Reaction, the PCR) technology of answering using its can detect the cause of disease DNA of trace and always by as examining
The golden mark method for a variety of epidemic diseases of breaking.But PCR need special heat circulating equipment, Cord blood reagent and avoid cross pollution
Technical operation requirement.Existing aviadenovirus detection method due to it is such or such the defects of and epidemic disease field diagnostic can not be met
Demand.Research and development is suitable for the on-site diagnosis technology of the type of aviadenovirus serum 4 for effectively control aviadenovirus blood
Chicken gizzard inflammation-hydropericardium syndrome caused by clear 4 type (FAdV-4) is significant.
Recombinase polymeric enzymatic amplification (Recombinase Polymerase Amplification, RPA) technology is by English
A kind of novel nucleic acids detection technique that can substitute normal PCR of state company's T wistDx Inc exploitations.RPA technologies rely primarily on
In three kinds of enzymes：Recombinase (Recombinase), the single strand binding protein (SSB) of single-chain nucleic acid (Oligonucleolide primers) can be combined
With strand displacement archaeal dna polymerase (Polymerase).The mixture of these three enzymes is also active at normal temperatures, and optimal reaction temperature exists
37 DEG C -42 DEG C or so.The Protein-DNA mixtures that recombinase combines to form with primer, can find homologous sequence in double-stranded DNA.
Once primer located homologous sequence, Exchange reaction of chain will occur and formed and start DNA synthesis, to the target area in template
Carry out exponential amplification.The DNA being replaced is combined with SSB, prevents from further replacing.It is relative by two in this system
Primer originate a compound event.Whole process carries out very fast, and can typically be obtained within ten minutes can detect level
Amplified production.Requirement of the technology to hardware device is very low, particularly suitable for field diagnostic, animal doctor, food security, biology
The fields such as safety, agricultural.RPA technologies have many advantages, such as different from Standard PCR：1) RPA reactions can be carried out at normal temperatures；
2) sensitivity of RPA detections is very high；3) it can be not only used for DNA, it can also be used to RNA amplification；4) multiplex amplification can be carried out.
Immune colloidal gold technique (Immune colloidal gold technique) is to be used as tracer label using collaurum
Thing is applied to a kind of new immunolabelling technique of antigen-antibody, and english abbreviation is：GICT.Collaurum is by gold chloride
(HAuCl4) under reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid effect, polymerization turns into the gold of particular size
Grain, and because electrostatic interaction turns into a kind of colloidal state of stabilization, referred to as collaurum.Collaurum is negatively charged under mild alkaline conditions
Lotus, firm combination can be formed with the positive charge group of protein molecule, because this combination is electrostatical binding, so not influenceing
The biological nature of protein.Collaurum can also be combined, such as in addition to being combined with protein with many other large biological molecules
SPA, PHA, ConA etc..According to some physical behaviors of collaurum, such as high electron density, granular size, shape and color reaction,
Plus the immune and biological characteristics of conjugate, thus collaurum is set to be widely used in immunology, histology, pathology and thin
The fields such as born of the same parents' biology.Colloidal gold immunity chromatography is to be fixed on specific antigen or antibody on film with ribbon, colloid
Golden labelled reagent (antibody or monoclonal antibody) absorption is on pad, when sample to be checked is added in the sample pad of test strips one end
Afterwards, moved forward, reacted to each other after dissolving the colloid gold label reagent on pad, then be moved to fixed by capillarity
During the region of antigen or antibody, the conjugate of thing and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
In detection band, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.
The existing method complex operation for being used to detect FAdV-4, expends time length, can not give clinic control by FAdV-4
Caused epidemic disease is provided and timely instructed, and therefore, there is an urgent need to find a kind of FAdV-4 field quick detections diagnostic techniques.
The content of the invention
The present invention is directed to problems of the prior art, there is provided a kind of RPA primers and detection for being used to detect FAdV-4
Method.The RPA primer specificities of the present invention are strong, and high sensitivity, association colloid gold immuno-chromatographic test paper strip detection technique, is effective
It is low, quick, special to detect a kind of chicken gizzard inflammation-cost of hydropericardium syndrome offer, and does not need the field diagnostic of special installation
New method.
To realize goal of the invention, the technical solution adopted by the present invention is as follows：
Present invention firstly provides a kind of RPA primers for being used to detect the type of aviadenovirus serum 4, wherein, the RPA primers
Nucleotides sequence be classified as：
Sense primer：5’-TTACACCCCTACCACGGACAACAGCGGACAGCAGCC-3’(SEQ ID NO.1)；
Anti-sense primer：5’-GATCTGTCAGTTCGCCCATGTACATAAAGTCGG-3’(SEQ ID NO.2).
According to above-mentioned RPA primers, it is preferable that 5 ' ends of the sense primer are carried out using fluorescein isothiocynate FITC
Mark, 5 ' ends of the anti-sense primer are marked using biotin.
Above-mentioned RPA primers can be used in preparing the reagent of the type of detection aviadenovirus serum 4.
Present invention also offers a kind of kit containing above-mentioned RPA primers, the kit can be used in detecting fowl adenopathy
The malicious type of serum 4.
Present invention also offers a kind of RPA methods for being used to detect the type of aviadenovirus serum 4, comprise the following steps：
(1) primer synthesizes：Synthesize the RPA primers described in claim 1 or 2；
(2) DNA profiling extracts：Extract the DNA in detected sample；
(3) RPA amplified reactions：Using the DNA of extraction in step (2) as template, using the RPA primers synthesized in step (1),
RPA amplified reactions are carried out in RPA reaction tubes；
(4) RPA amplified productions are analyzed.
According to above-mentioned RPA methods, it is preferable that using TwistDx Inc companies of BritainReagent
Box carries out RPA amplified reactions, and the RPA amplification reaction systems are calculated as with 50 μ l：The μ l of rehydration buffer 29.5, magnesium acetate are (initial
Concentration is 280mM) 2.5 μ l, and primer, template DNA and the nuclease free pure water mixture that cumulative volume is 18 μ l.The 18 μ l
Primer, template DNA and nuclease free pure water mixture be specially：(initial concentration is 0.25 μ for sense primer and anti-sense primer
M) each 2 μ l, the μ l of template DNA 2, the μ l of nuclease free pure water 12.
According to above-mentioned RPA methods, the Loading sequence and reaction condition of the RPA amplified reactions are：RPA is reacted first
Composition in system in addition to magnesium acetate is added toThe lyophilized multienzyme complex reaction tube that kit provides
In, then magnesium acetate is added to the cover inner surface of lyophilized multienzyme complex reaction tube, close the lid, brief centrifugation, make acetic acid
Magnesium, which enters, starts reaction in reaction system；Then lyophilized multienzyme complex reaction tube is put intoReactor or thermostatted water
In bath or the thermostat of other forms, 37 DEG C of constant-temperature incubations 30 minutes.
According to above-mentioned RPA methods, the method for analysis RPA amplified productions is in step (4)：RPA amplified productions are diluted,
Then the RPA amplified productions after dilution are detected with collaurum lateral flow immunochromatography test strips.Preferably, the colloid
Coloured particle in golden lateral flow immunochromatography test strips is nanogold particle, and the nanogold particle uses Streptavidin bag
Quilt.Preferably, the tunica fibrosa in the collaurum lateral flow immunochromatography test strips is nitrocellulose filter.
According to above-mentioned RPA methods, it is preferable that utilize the analysis RPA amplification productions of collaurum lateral flow immunochromatography test strips
The method of thing is specially：5 μ l RPA amplified matters are added and fill 95 μ l Tris salt buffers (25mM Tris, 150mM NaCl
And 0.05%Tween-20) test tube in, be diluted, then by the coupling pads of collaurum lateral flow immunochromatography test strips end
End is inserted perpendicularly into test tube, makes coupling pad end contact solution.After 10 minutes, take out test strips and observed or put down with suitable
Plate scanner is scanned.
According to above-mentioned RPA methods, the collaurum lateral flow immunochromatography test strips are provided with anti-isosulfocyanic acid fluorescence
Plain antibody detection line and biotinylated antibody nature controlling line；If the anti-isosulfocyanic acid fluorescence of collaurum lateral flow immunochromatography test strips
There is band in plain antibody detection line, and biotinylated antibody nature controlling line is normal, then shows to contain aviadenovirus blood in the sample
Clear 4 type, if being only band occur on biotinylated antibody nature controlling line, show to be free of the type of aviadenovirus serum 4 in the sample.
In addition it is also possible to analyze RPA amplified productions with agarose gel electrophoresis, in the case of target gene presence, RPA is anti-
The fragment expanded by upstream and downstream primer should be produced, suitable primer can be screened by way of gel electrophoresis, to ensure
It is able to detect that purpose product.
The present invention principle be：Present invention design amplification FAdV-4 specific primer, can detect FAdV-4 presence.
The primer of specific amplification FAdV-4 fragments is marked using label, after RPA reacts, the final one end that produces carries
Biotin, the other end carry the double labelling product of fluorescein isothiocynate (FITC), and the double labelling product, which is coated, has strepto- close
Adsorbed with the red nano gold microsphere of element, nanometer gold microsphere drives to be moved in the lateral flow power of liquid is fixed on examination in advance
Anti- fluorescein isothiocynate antibody and biotinylated antibody position on paper slip, gradual deposition form macroscopic
Precipitation line, it can utilize the detection line occurred in test strips whereby and reach detection FAdV-4 purpose.
The positive beneficial effect that the present invention obtains：
(1) the RPA primer specificities that the present invention designs are strong, high sensitivity, and testing result is accurate.
(2) detection method of the invention joins the normal temperature RPA technologies for detecting FAdV-4 with colloidal gold immuno-chromatography test paper strip
With can realize FAdV-4 quick detection, can at least detect the FAdV-4 of 150 copies.The detection method specificity of the present invention
Result of the test is good, and replica test has good stability, and one is provided for effective detection chicken gizzard inflammation-hydropericardium syndrome
Kind cost is low, quick, special, it is not necessary to the field diagnostic new method of special installation, is advantageous to chicken gizzard inflammation-hydropericardium syndrome
Antidiastole, while high instrument input can also be exempted, be easy to basic unit to promote the use of.
(3) present invention can as feeding chicken in largely scale field and free-range farm FAdV-4 rapid field detection method, also for
The Molecule Epidemiology Investigation of FAdV-4 infection, development and the exploitation of diagnosis test paper provide test basis and Technical Reference.
Brief description of the drawings
Fig. 1 is collaurum lateral flow immunochromatography test strips building block principle schematic diagram of the present invention；
Fig. 2 is collaurum lateral flow immunochromatography test strips operation principle schematic diagram of the present invention；
Fig. 3 is the ELISA test strip result of RPA primer specificities of the present invention detection；
Fig. 4 is the ELISA test strip result of RPA primers sensitivity technique of the present invention.
Embodiment
The details and form of technical solution of the present invention can be modified without departing from the spirit and scope of the invention
Or replace, but these modifications or substitutions each fall within protection scope of the present invention.Unless otherwise specified, original used in embodiment
Material, chemical reagent are conventional commercial commodity, technological means used conventional meanses known to those skilled in the art.
It is using RPA kits in following examplesKit, it is public purchased from Britain TwistDx Inc
Department；Wherein, recombinase, single-stranded DNA binding protein (SSB) and the strand displacement DNA of single-chain nucleic acid (Oligonucleolide primers) can be combined
Polymerase is present in freeze-dried powder state in the RPA reaction tubes that kit is provided, directly with kit institute band during use
Reaction buffer dilutes.Carried out during whole RPA reactions in RPA reaction tubes.
Embodiment 1：For the synthesis for the primer for detecting the type of aviadenovirus serum 4
According to the FAdV-4 gene order comparison results announced in GenBank, FAdV-4 genome nucleotide sequences are selected
The sequence (5 '-FITC-TTACACCCCTACCACGGACAACAGCGGACAGCAGCC-3 ') of 2355bp-2390bp positions in row
As the sense primer of RPA amplified reactions, in its 5 ' end mark fluorescein isothiocynate (FITC) when synthesizing sense primer；With
The reverse complementary sequence of 2620bp-2652bp position sequences is as under RPA amplified reactions in FAdV-4 genome nucleotide sequences
Primer is swum, in its 5 ' end mark biotins (Biotin) when synthesizing anti-sense primer：(5’-Biotin-
GATCTGTCAGTTCGCCCATGTACATAAAGTCGG-3’)。
Primer synthesis is completed by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 2：The preparation of collaurum lateral flow immunochromatography test strips
(1) preparation of the coated nanogold particle of Streptavidin：1ml gold nano grains solution (0.15pmol/mL) is taken,
Add 200mM borax soln, adjustment pH value to 6.5.Meanwhile by 2 μ l Streptavidin (2mg/ in another test tube
Ml) mixed with 398 μ l borax soln (2mM)；The Streptavidin of dilution is added in foregoing nanogold particle solution,
Progressively increased with 50 μ l amount, it is stirring while adding.Mixture is put into room temperature to place 45 minutes, added containing 155.6 μ l containing 10%BSA's
2mM borax solns, continue in room temperature to place solution 10 minutes, 4500g is centrifuged 15 minutes, and liquid is abandoned in suction, with 1ml cleaning solution
Precipitation is resuspended in (the 2mM borax solns containing 10g/L BSA), and 4500g is centrifuged 15 minutes, and liquid is abandoned in suction, by the precipitation weight of red
250 μ l are suspended in contain in the buffer solution of 5%BSA, 137mM NaCl and 0.025% Tween-20.Prepared according to this ratio enough
The coated nanogold particle of Streptavidin, take the above-mentioned coated nanogold particles of 250 μ l to be added drop-wise to 7mm × 300mm laser and cut
On the fiberglass packing cut, it is allowed to diffusion uniformly, is dried overnight on experimental bench.
(2) preparation of collaurum lateral flow immunochromatography test strips：With the 100mM carbonic acid containing 5% methanol, 2% sucrose
Anti- fluorescein isothiocynate antibody and biotinylated anti-mouse IgG are diluted to final concentration of 0.5mg/ml by hydrogen sodium buffer solution respectively
And 1.0mg/ml.All antibody use lateral flow reagent distributor, and the speed of dispenser head is set in 1~3 cm per minute, excellent
Elect 2 cm per minutes as, the flow rate set of syringe pump is in 0.1~0.3ml/ minutes, preferably 0.1ml/ minutes.When two kinds of antibody
In test strips after the completion of point sample, test strips are dried 1 hour at 37 DEG C.Then test card is carried out according to Fig. 2 and following steps
Assembling：1. one 17 millimeters × 300 millimeters of absorption pad is placed on to the right hand downstream of the nitrocellulose filter of plastic support,
The two overlapping 2 millimeters；2. it is placed on nitric acid fibre containing the fiberglass packing for drying gold nano grain by one 7 millimeters × 300 millimeters
The left hand upstream end of plain film is tieed up, the two overlapping 2 millimeters；3. one 12 millimeters × 300 millimeters of glass fiber sample pad is placed on
The left-hand end of gold nano grain pad, the two overlapping 2 millimeters.After the completion of assembling, test card is cut into the test strips of 3 mm wides immediately
Obtain collaurum lateral flow immunochromatography test strips, sealing, kept dry.
Embodiment 3：DNA is extracted
The type DNA of aviadenovirus serum 4 extraction：Utilize the TIANamp Genomic DNA Kit reagents of TIANGEN companies
Box extracts STb gene, and concrete operations are as follows：1. taking 200 μ l FAdV-4 cell toxicants in 1.5ml eppendorf pipes, egg is added
The white μ l (concentration 20mg/ml) of enzyme K 20；2. often pipe adds 200 μ l buffer solution GB, fully reverse to mix, 70 DEG C of placement 10min,
Solution becomes limpid rear brief centrifugation to remove the globule in pipe；3. adding 200 μ l absolute ethyl alcohols, fully shaking mixes, brief centrifugation
To remove the globule in pipe；4. the solution obtained by previous step and flocculent deposit are all added in an adsorption column CB3, (adsorption column is put
Enter into collecting pipe), 12000rpm centrifugation 30s, waste liquid is outwelled, adsorption column CB3 is put back in collecting pipe；5. to adsorption column CB3
It is middle to add 500 μ l buffer solutions GD, 12000rpm centrifugation 30s, waste liquid is outwelled, adsorption column CB3 is put into collecting pipe；6. to absorption
600 μ l rinsing liquids PW, 12000rpm centrifugation 30s are added in post CB3, waste liquid is outwelled, adsorption column CB3 is put into collecting pipe
In；7. repeat step is 6.；8. adsorption column CB3 is put back in collecting pipe, 12000rpm centrifugation 2min, waste liquid is outwelled.By adsorption column
CB3 is placed in room temperature and placed several minutes, thoroughly to dry rinsing liquid remaining in adsorption column；9. adsorption column CB3 is transferred into one to do
In net centrifuge tube, think that 100 μ l elution buffer TE are vacantly added dropwise in the middle part of adsorbed film, room temperature places 2-5min,
12000rpm centrifuges 2min, solution is collected into centrifuge tube, that is, the genomic DNA of the type of aviadenovirus serum 4 extracted,
Be stored in -20 DEG C it is standby.
Embodiment 4：Primer specificity is tested
Respectively with the type of aviadenovirus serum 4 (FAdV-4), aviadenovirus serum 8b types (FAdV-8b), egg drop syndrome disease
Malicious (Egg drop syndrome virus, EDSV), Marek's disease virus (Marek ' s disease virus, MDV) and biography
The DNA of metachromia laryngotracheitis virus (Infectious Laryngotracheitis virus, ILTV), NDV
(Newcastle disease virus, NDV), avian influenza virus H9 hypotypes (Avian influenza virus
H9subtype, AIV-H9), infectious bursal disease virus ((Infectious bursal disease virus, IBDV) and
The cDNA of infectious bronchitis virus (Infectious bronchitis virus, IBV) is template, using Britain
TwistDx Inc companiesKit carries out RPA amplified reactions, and the primer synthesized in embodiment 1 is entered
Row specificity verification.
The RPA amplification reaction systems are calculated as with 50 μ l：29.5 μ l rehydration buffers, μ l of template DNA 2, upstream and downstream
Primer (initial concentration is 0.25 μM) each 2 μ l, the μ l of nuclease free pure water 12 and the μ l of magnesium acetate (concentration 280mM) 2.5.
The Loading sequence and reaction condition of the RPA amplified reactions be：First by RPA reaction systems except magnesium acetate it
Outer composition is added toIn the lyophilized multienzyme complex reaction tube that kit provides, then magnesium acetate is added
Enter the cover inner surface to lyophilized multienzyme complex reaction tube, then close the lid, brief centrifugation, magnesium acetate is entered reaction system
It is middle to start reaction；Lyophilized multienzyme complex reaction tube is put intoThe perseverance of reactor or thermostat water bath or other forms
In warm device, 37 DEG C of constant-temperature incubations 30 minutes.
After incubation terminates, 5 μ l RPA amplified matters are taken, are containing 95 μ l Tris salt buffers (25mMTris, 150mMNaCl
And 0.05%Tween-20) test tube in dilute, the couplings of collaurum lateral flow immunochromatography test strips pad ending vertical is inserted
Enter in test tube, make coupling pad end contact solution.After 10 minutes, take out test strips and observed or entered with suitable flat bed scanner
Row scanning.As a result (Fig. 3) is shown, the primer for the type of aviadenovirus serum 4 designed by the present invention has good specificity.
Embodiment 5：Primer sensitivity analysis is tested
The DNA extracted with embodiment 3, using TwistDx Inc companies of BritainBasic kits are carried out
RPA amplified reactions, to detect the sensitivity of the primer synthesized in embodiment 1.Specifically test operation is：By aviadenovirus serum 4
The DNA of type carries out 10 times of continuous doubling dilutions, carries out RPA amplified reactions by template of the DNA of the various concentrations after dilution respectively,
To determine the sensitivity of primer.RPA amplification reaction systems, Loading sequence and the reaction condition are the same as embodiment 4.Wherein, DNA is copied
Shellfish number is calculated as：The OD260nm absorbances determined using ultraviolet specrophotometer, according to formula DNA concentration=OD260 value × 50
μ g/ml calculate DNA concentration, according to formula (6.02 × 1023Copy number/mole) × (DNA concentration × 10-9)/(DNA length ×
660)=DNA copy number (copies/ul) calculates DNA copy number.
After RPA amplified reactions terminate, take 5 μ l RPA amplified matters, containing 95 μ lTris salt buffers (25mM Tris,
150mM NaCl and 0.05%Tween-20) test tube in dilute, by the coupling pad of collaurum lateral flow immunochromatography test strips
In ending vertical insertion test tube, make coupling pad end contact solution.After 10 minutes, take out test strips and observed or put down with suitable
Plate scanner is scanned.As a result (Fig. 4) is shown, the primer and established detection method designed by the present invention can be detected at least
The FAdV-4 of 150 copies.
Embodiment 6：The optimization of the type reaction condition of RPA amplified reactions detection aviadenovirus serum 4
Using the DNA of the type of aviadenovirus serum 4 as template, using the Twist of TwistDx Inc companies of Britain
Kit carries out RPA amplified reactions, to the primer concentration in RPA amplification reaction systems, acetic acid magnesium density, template concentrations, reaction
Temperature (37-42 DEG C) and reaction time (15-45min) optimize.The RPA reaction systems are 50 μ l, and main component includes
29.5 μ l rehydration buffers, 0.5-2 μ l template DNAs, sense primer (initial concentration is 0.25 μM) 1.0-3.0 μ l, anti-sense primer
(initial concentration is 0.25 μM) 1.0-3.0 μ l and 1.0-2.5 μ l magnesium acetates (initial concentration 280mM), with nuclease free pure water
Reaction volume is supplied to 50 μ l.
The Loading sequence and reaction condition of the RPA amplified reactions be：First by RPA reaction systems except magnesium acetate it
Outer composition is added toIn the lyophilized multienzyme complex reaction tube that kit provides, then magnesium acetate is added
Enter the cover inner surface to lyophilized multienzyme complex reaction tube, then close the lid, brief centrifugation, magnesium acetate is entered reaction system
It is middle to start reaction；Lyophilized multienzyme complex reaction tube is put intoThe perseverance of reactor or thermostat water bath or other forms
In warm device, 37 DEG C of -42 DEG C of constant-temperature incubation 15-45 minutes.
After incubation terminates, 5 μ l RPA amplified matters are taken, are containing 95 μ l Tris salt buffers (25mM Tris, 150mM
NaCl and 0.05%Tween-20) test tube in dilute, the couplings of collaurum lateral flow immunochromatography test strips pad end is hung down
Straight cutting enters in test tube, makes coupling pad end contact solution.After 10 minutes, take out test strips and observed or with suitable flat-bed scanning
Instrument is scanned.
By the optimization to RPA amplification reaction conditions, the RPA for finally giving the type of detection aviadenovirus serum 4 reacts excellent
Change condition, its optimal conditions are：Sense primer (initial concentration is 0.25 μM) 2 μ l, anti-sense primer (initial concentration is 0.25 μM) 2
μ l, the μ l of template DNA 2, the μ l of magnesium acetate (initial concentration 280mM) 2.5, reaction temperature are 37 DEG C, reaction time 30min.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of He'nan
<120>A kind of RPA primers and its detection method for being used to detect the type of aviadenovirus serum 4
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 1
ttacacccct accacggaca acagcggaca gcagcc 36
<210> 2
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 2
gatctgtcag ttcgcccatg tacataaagt cgg 33
Claims (10)
- A kind of 1. RPA primers for being used to detect the type of aviadenovirus serum 4, it is characterised in that the nucleotide sequence of the RPA primers For：Sense primer：5’-TTACACCCCTACCACGGACAACAGCGGACAGCAGCC-3’(SEQ ID NO.1)；Anti-sense primer：5’-GATCTGTCAGTTCGCCCATGTACATAAAGTCGG-3’(SEQ ID NO.2).
- 2. RPA primers according to claim 1, it is characterised in that 5 ' ends of the sense primer are glimmering using isothiocyanic acid Light element FITC is marked, and 5 ' ends of the anti-sense primer are marked using biotin.
- 3. application of the RPA primers in the type reagent of detection aviadenovirus serum 4 is prepared described in claim 1 or 2.
- 4. the kit containing the RPA primers described in claim 1 or 2.
- 5. application of the kit in the type of aviadenovirus serum 4 is detected described in claim 4.
- A kind of 6. RPA methods for being used to detect the type of aviadenovirus serum 4, it is characterised in that comprise the following steps：(1) primer synthesizes：Synthesize the RPA primers described in claim 1 or 2；(2) DNA profiling extracts：Extract the DNA in detected sample；(3) RPA amplified reactions：Using the DNA of extraction in step (2) as template, using the RPA primers synthesized in step (1), RPA amplified reactions are carried out in RPA reaction tubes；(4) RPA amplified productions are analyzed.
- 7. RPA methods according to claim 6, it is characterised in that the RPA amplification reaction systems are calculated as with 50 μ l：Wherein, the initial concentration of sense primer and anti-sense primer is 0.25 μM, and the initial concentration of the magnesium acetate is 280mM.
- 8. RPA methods according to claim 6, it is characterised in that the method for analysis RPA amplified productions is in step (4)： RPA amplified productions are diluted, then the RPA amplified productions after dilution examined with collaurum lateral flow immunochromatography test strips Survey.
- 9. RPA methods according to claim 8, it is characterised in that in the collaurum lateral flow immunochromatography test strips Coloured particle be nanogold particle, the nanogold particle using Streptavidin be coated with.
- 10. RPA methods according to claim 8, it is characterised in that in the collaurum lateral flow immunochromatography test strips Provided with anti-fluorescein isothiocynate antibody detection line and biotinylated antibody nature controlling line；If collaurum lateral flow immunochromatography test paper There is band in the anti-fluorescein isothiocynate antibody detection line of bar, and biotinylated antibody nature controlling line is normal, then shows the sample Contain the type of aviadenovirus serum 4 in product, if being only band occur on biotinylated antibody nature controlling line, show to be free of in the sample The type of aviadenovirus serum 4.
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