CN107385110B - A kind of RPA primers and its detection method for detecting 4 type of aviadenovirus serum - Google Patents
A kind of RPA primers and its detection method for detecting 4 type of aviadenovirus serum Download PDFInfo
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- 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
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- 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
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- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of RPA primers for detecting 4 type of aviadenovirus serum, and 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 detecting 4 type of aviadenovirus serum, this method includes mainly：Primer synthesis, sample to be tested DNA extractions, RPA amplified reactions and the analysis of RPA amplified productions and etc..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 easy to operate, and stability is good, and a kind of field diagnostic method at low cost, quick, special is provided for effective detection and identification chicken gizzard inflammation-hydropericardium syndrome.
Description
Technical field
The invention belongs to molecular Biological Detection fields, and in particular to the RPA primers for detecting 4 type of aviadenovirus serum
And its detection method.
Background technology
Chicken gizzard inflammation caused by 4 type of aviadenovirus serum (Fowl adenovirus serotype4, FAdV-4)-pericardium product
Liquid syndrome is highly contagious disease, which 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 prodigious 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 the maximum 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 sensibility of agar gel diffusion test is not high enough；ELISA is usually used
It is complex for operation step in the antibody response for measuring specific adenovirus, while being also required to specific instrument；Conventional polymerase chain is anti-
Answer (Polymerase Chain Reaction, PCR) technology using it 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 contamination
Technical operation requirement.Existing aviadenovirus detection method cannot be satisfied epidemic disease field diagnostic due to such or such defect
Demand.Research and development is suitable for the on-site diagnosis technology of 4 type of aviadenovirus serum for effectively controlling aviadenovirus blood
Chicken gizzard inflammation-hydropericardium syndrome is of great significance caused by clear 4 type (FAdV-4).
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.Recombinase is combined the Protein-DNA mixtures to be formed with primer, and homologous sequence can be found 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 chain being replaced is combined with SSB, prevents from further replacing.It is opposite by two in this system
Primer originate a compound event.Whole process carries out very fast, and can generally 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 carry 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 the amplification of RNA；4) multiplex amplification can be carried out.
Immune colloidal gold technique (Immune colloidal gold technique) is using colloidal gold as tracer label
Object is applied to a kind of novel immunolabelling technique of antigen-antibody, and english abbreviation is：GICT.Colloidal gold is by gold chloride
(HAuCl4) under the reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid the effects that, polymerization as particular size gold
Grain, and since electrostatic interaction becomes a kind of colloidal state of stabilization, referred to as colloidal gold.Colloidal gold is negatively charged under mild alkaline conditions
Lotus can form firm combination, since this combination is electrostatical binding, so not influencing with the positive charge group of protein molecule
The biological nature of protein.Colloidal gold can also be combined, such as other than being combined with protein with many other large biological molecules
SPA, PHA, ConA etc..According to some physical behaviors of colloidal gold, such as high electron density, granular size, shape and color reaction,
In addition the immune and biological characteristics of conjugate, thus colloidal gold is made 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 the antigen of specificity or antibody on film with ribbon, colloid
Golden labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be checked is added in the sample pad of test strips one end
Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad, then be moved to fixed through capillary action
When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
It is taken in detection, 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 for detecting FAdV-4 is complicated for operation, and the consuming time is long, clinic control can not be given by FAdV-4
Caused epidemic disease offer is timely instructed, and therefore, there is an urgent need to find a kind of FAdV-4 field quick detections diagnostic techniques.
Invention content
The present invention is directed to problems of the prior art, provides a kind of RPA primers and detection for detecting 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 a kind of at low cost, quick, special to detect chicken gizzard inflammation-hydropericardium syndrome offer, and does not need the field diagnostic of special installation
New method.
To realize that goal of the invention, the technical solution adopted by the present invention are as follows：
Present invention firstly provides a kind of RPA primers for detecting 4 type of aviadenovirus serum, wherein the RPA primers
Nucleotides sequence be classified as：
Sense primer：5'-TTACACCCCTACCACGGACAACAGCGGACAGCAGCC-3'(SEQ ID NO.1)；
Downstream 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
5 ' ends of label, the downstream primer are marked using biotin.
Above-mentioned RPA primers can be used in preparing the reagent of 4 type of detection aviadenovirus serum.
The present invention also provides a kind of kit containing above-mentioned RPA primers, which can be used in detecting fowl adenopathy
4 type of malicious serum.
The present invention also provides a kind of RPA methods for detecting 4 type of aviadenovirus serum, include the following steps：
(1) primer synthesizes：Synthesize RPA primers as claimed in claim 1 or 2；
(2) DNA profiling extracts：Extract the DNA in detected sample；
(3) RPA amplified reactions：Using the DNA extracted in step (2) as template, using the RPA primers of synthesis 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：29.5 μ l of rehydration buffer, magnesium acetate are (initial
A concentration of 280mM) 2.5 μ l and total volume be 18 μ l primer, template DNA and nuclease free pure water mixture.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 downstream primer
M) each 2 μ l, 2 μ l of template DNA, 12 μ l of nuclease free pure water.
According to above-mentioned RPA methods, the Loading sequence and reaction condition of the RPA amplified reactions are：RPA is reacted first
Ingredient in system other than magnesium acetate is added toThe freeze-drying multienzyme complex reaction tube that kit provides
In, then magnesium acetate is added to the cover inner surface of freeze-drying multienzyme complex reaction tube, closed the lid, brief centrifugation makes acetic acid
Magnesium, which enters, starts reaction in reaction system；Then freeze-drying 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 colloidal gold 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 packet
Quilt.Preferably, the tunica fibrosa in the colloidal gold 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 colloidal gold lateral flow immunochromatography test strips
The method of object is specially：The addition of 5 μ l RPA amplified matters is filled into 95 μ l Tris salt buffers (25mM Tris, 150mM NaCl
And 0.05%Tween-20) test tube in, be diluted, then by the coupling pad of colloidal gold lateral flow immunochromatography test strips end
End is inserted perpendicularly into test tube, makes coupling pad end contact solution.After ten minutes, test strips are taken out to carry out observation or put down with suitable
Plate scanner is scanned.
According to above-mentioned RPA methods, the colloidal gold lateral flow immunochromatography test strips are equipped with anti-isosulfocyanic acid fluorescence
Plain antibody detection line and biotinylated antibody nature controlling line；If the anti-isosulfocyanic acid fluorescence of colloidal gold 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 shows to be free of 4 type of aviadenovirus serum in the sample if being only band occur on biotinylated antibody nature controlling line.
In addition it is also possible to analyze RPA amplified productions with agarose gel electrophoresis, exist in target gene, RPA is anti-
It should generate by the segment of upstream and downstream primer amplification, suitable primer can be screened by way of gel electrophoresis, to ensure
It is able to detect that purpose product.
The principle of the present invention is：The present invention designs the specific primer of amplification FAdV-4, can detect the presence of FAdV-4.
The primer of specific amplification FAdV-4 segments is marked using marker, after RPA reacts, the final one end that generates carries
Biotin, the other end carry the double labelling product of fluorescein isothiocynate (FITC), which, which is coated, has strepto- close
It is adsorbed with the red nano gold microsphere of element, nanometer gold microsphere is moved in the lateral flow power drive of liquid and is fixed on examination in advance
Anti- fluorescein isothiocynate antibody on paper slip and biotinylated antibody position, gradual deposition form macroscopic
Precipitation line can achieve the purpose that detect FAdV-4 whereby using the detection line occurred in test strips.
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 room temperature RPA technologies for detecting FAdV-4 with colloidal gold immuno-chromatography test paper strip
With can realize the quick detection of FAdV-4, can at least detect the FAdV-4 of 150 copies.The detection method specificity of the present invention
Test result is good, and repetitive test has good stability, and one is provided for effectively detection chicken gizzard inflammation-hydropericardium syndrome
Kind is at low cost, quick, special, does not need the field diagnostic new method of special installation, is conducive to chicken gizzard inflammation-hydropericardium syndrome
Antidiastole, while high instrument input can also be exempted, be promoted the use of convenient for base.
(3) present invention can as the rapid field detection method of feeding chicken in largely scale field and free-range farm FAdV-4, also for
The Molecule Epidemiology Investigation of FAdV-4 infection, development and the exploitation of diagnosis test paper provide test basis and Technical Reference.
Description of the drawings
Fig. 1 is colloidal gold lateral flow immunochromatography test strips building block principle schematic diagram of the present invention；
Fig. 2 is colloidal gold 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.
Specific implementation mode
It can modify without departing from the spirit and scope of the invention to the details and form of technical solution of the present invention
Or replace, but these modifications or substitutions each fall within protection scope of the present invention.Unless otherwise specified, former used in embodiment
Material, chemical reagent are conventional commercial commodity, technological means used conventional means known to those skilled in the art.
It is using RPA kits in following embodimentKit, it is public purchased from Britain TwistDx Inc
Department；It wherein, can be in conjunction with recombinase, single-stranded DNA binding protein (SSB) and the strand displacement DNA of single-chain nucleic acid (Oligonucleolide primers)
Polymerase is present in freeze-dried powder state in the RPA reaction tubes that kit is provided, and kit institute band is directly used when use
Reaction buffer dilutes.It is carried out in RPA reaction tubes when entire RPA reactions.
Embodiment 1：Synthesis for the primer for detecting 4 type of aviadenovirus serum
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 the positions 2355bp-2390bp in row
As the sense primer of RPA amplified reactions, in its 5 ' end label 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 label biotins (Biotin) when synthesizing downstream primer：(5'-Biotin-
GATCTGTCAGTTCGCCCATGTACATAAAGTCGG-3’)。
Primer synthesis is completed by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 2：The preparation of colloidal gold lateral flow immunochromatography test strips
(1) preparation of the coated nanogold particle of Streptavidin：1ml gold nano grains solution (0.15pmol/mL) is taken,
The borax soln of 200mM, adjustment pH value to 6.5 is added.Meanwhile by the Streptavidin (2mg/ of 2 μ l in another test tube
Ml it) is mixed with the borax soln of 398 μ l (2mM)；Diluted Streptavidin is added in nanogold particle solution above-mentioned,
Progressively increased with the amount of 50 μ l, it is stirring while adding.Mixture is set and is placed at room temperature for 45 minutes, is added containing 155.6 μ l containing 10%BSA's
2mM borax solns continue solution being placed at room temperature for 10 minutes, and 4500g is centrifuged 15 minutes, and liquid is abandoned in suction, with the cleaning solution of 1ml
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 red precipitation weight
It is suspended in the buffer solution that 250 μ l contain 5%BSA, 137mM NaCl and 0.025% Tween-20.It is prepared according to this ratio enough
The coated nanogold particle of Streptavidin takes the above-mentioned coated nanogold particles of 250 μ l to be added drop-wise to 7mm × 300mm laser and cuts
On the fiberglass packing cut, it is allowed to diffusion uniformly, is dried overnight on experimental bench.
(2) preparation of colloidal gold 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
2 cm per minutes are selected as, the flow rate set of syringe pump was at 0.1~0.3ml/ minutes, preferably 0.1ml/ minutes.When two kinds of antibody
It is after the completion of point sample in test strips, test strips are 1 hour dry at 37 DEG C.Then test card is carried out according to Fig. 2 and following steps
Assembly：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 is overlapped 2 millimeters；2. one 7 millimeters × 300 millimeters fiberglass packings containing dry gold nano grain are placed on nitric acid fibre
The left hand upstream end of the plain film of dimension, the two are overlapped 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 are overlapped 2 millimeters.After the completion of assembly, test card is cut into the test strips of 3 mm wides immediately
Obtain colloidal gold lateral flow immunochromatography test strips, sealing, kept dry.
Embodiment 3：DNA is extracted
The extraction of 4 type DNA of aviadenovirus serum：Utilize the TIANamp Genomic DNA Kit reagents of TIANGEN companies
Box extracts total DNA, and concrete operations are as follows：1. taking 200 μ l FAdV-4 cell toxicants in the eppendorf pipes of 1.5ml, egg is added
White 20 μ l (a concentration of 20mg/ml) of enzyme K；2. often 200 μ l buffer solution GB are added in pipe, fully reverse mixing, 70 DEG C of placement 10min,
Brief centrifugation is to remove droplet in pipe after solution becomes limpid；3. 200 μ l absolute ethyl alcohols, fully shaking mixing, brief centrifugation is added
Interior droplet is managed with removal；4. by obtained by previous step solution and flocculent deposit be all added in an adsorption column CB3 that (adsorption column is put
Enter into collecting pipe), 12000rpm centrifuges 30s, outwells waste liquid, adsorption column CB3 is put back in collecting pipe；5. to adsorption column CB3
It is middle that 500 μ l buffer solutions GD, 12000rpm centrifugation 30s are added, 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 column CB3, outwells waste liquid, adsorption column CB3 is put into collecting pipe
In；7. repeating step 6.；8. adsorption column CB3 is put back in collecting pipe, 12000rpm centrifuges 2min, outwells waste liquid.By adsorption column
CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorption column；9. adsorption column CB3 is transferred to one to do
In net centrifuge tube, thinks that 100 μ l elution buffer TE are vacantly added dropwise in the intermediate position of adsorbed film, is placed at room temperature for 2-5min,
12000rpm centrifuges 2min, and solution is collected into centrifuge tube to get to the genomic DNA of 4 type of aviadenovirus serum of extraction,
Be stored in -20 DEG C it is spare.
Embodiment 4：Primer specificity is tested
Respectively with 4 type of aviadenovirus serum (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), newcastle disease virus
(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, to the primer that is synthesized in embodiment 1 into
Row specificity verification.
The RPA amplification reaction systems are calculated as with 50 μ l：29.5 μ l rehydration buffers, 2 μ l of template DNA, upstream and downstream
Primer (initial concentration is 0.25 μM) each 2 μ l, 12 μ l of nuclease free pure water and 2.5 μ l of magnesium acetate (a concentration of 280mM).
The Loading sequence and reaction condition of the RPA amplified reactions be：First by RPA reaction systems in addition to magnesium acetate it
Outer ingredient is added toIn the freeze-drying multienzyme complex reaction tube that kit provides, then magnesium acetate is added
Enter the cover inner surface to freeze-drying multienzyme complex reaction tube, then closes the lid, brief centrifugation makes magnesium acetate enter reaction system
Middle startup reaction；Freeze-drying 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, 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 colloidal gold lateral flow immunochromatography test strips pad ending vertical is inserted
Enter in test tube, makes coupling pad end contact solution.After ten minutes, take out test strips carry out observation or with suitable flat bed scanner into
Row scanning.As a result (Fig. 3) is shown, the primer for 4 type of aviadenovirus serum designed by the present invention has good specificity.
Embodiment 5：Primer sensitivity analysis is tested
With the DNA that embodiment 3 is extracted, using TwistDx Inc companies of BritainBasic kits carry 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 concentration after dilution respectively,
To determine the sensitivity of primer.The RPA amplification reaction systems, Loading sequence and reaction condition are the same as embodiment 4.Wherein, DNA is copied
Shellfish number is calculated as：The OD260nm absorbances measured using ultraviolet specrophotometer, according to formula DNA concentration=value × 50 OD260
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 the copy number of DNA.
After RPA amplified reactions, 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 colloidal gold lateral flow immunochromatography test strips
Ending vertical is inserted into test tube, makes coupling pad end contact solution.After ten minutes, test strips are taken out to carry out observation 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：RPA amplified reactions detect the optimization of 4 type reaction condition of aviadenovirus serum
Using the DNA of 4 type of aviadenovirus serum 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, downstream 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 in addition to magnesium acetate it
Outer ingredient is added toIn the freeze-drying multienzyme complex reaction tube that kit provides, then magnesium acetate is added
Enter the cover inner surface to freeze-drying multienzyme complex reaction tube, then closes the lid, brief centrifugation makes magnesium acetate enter reaction system
Middle startup reaction；Freeze-drying 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 incubations 15-45 minutes.
After incubation, 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 colloidal gold lateral flow immunochromatography test strips pad end is hung down
Straight cutting enters in test tube, makes coupling pad end contact solution.After ten minutes, test strips are taken out and carry out observation or with suitable flat-bed scanning
Instrument is scanned.
By the optimization to RPA amplification reaction conditions, the excellent of the RPA reactions of 4 type of detection aviadenovirus serum is finally obtained
Change condition, optimal conditions are：Sense primer (initial concentration is 0.25 μM) 2 μ l, downstream primer (initial concentration is 0.25 μM) 2
μ l, 2 μ l of template DNA, 2.5 μ l of magnesium acetate (initial concentration 280mM), 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 detecting 4 type of aviadenovirus serum
<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)
1. a kind of RPA primers for detecting 4 type of aviadenovirus serum, which is characterized in that the nucleotide sequence of the RPA primers
For：
Sense primer：5'-TTACACCCCTACCACGGACAACAGCGGACAGCAGCC-3'（SEQ ID NO.1）；
Downstream primer：5'-GATCTGTCAGTTCGCCCATGTACATAAAGTCGG-3'（SEQ ID NO.2）.
2. RPA primers according to claim 1, which is characterized in that 5 ' ends of the sense primer are glimmering using isothiocyanic acid
Light element FITC is marked, and 5 ' ends of the downstream primer are marked using biotin.
3. application of the RPA primers as claimed in claim 1 or 2 in preparing 4 type reagent of detection aviadenovirus serum.
4. the kit containing RPA primers as claimed in claim 1 or 2.
5. application of the kit in detecting 4 type of aviadenovirus serum described in claim 4, the application is not with disease detection
And/or for the purpose for the treatment of.
6. a kind of RPA methods for detecting 4 type of aviadenovirus serum, which is characterized in that include the following steps：
（1）Primer synthesizes：Synthesize the RPA primers described in claim 2；
（2）DNA template extractions：Extract the DNA in detected sample；
（3）RPA amplified reactions：With step（2）The DNA of middle extraction is template, using step（1）The RPA primers of middle synthesis,
RPA amplified reactions are carried out in RPA reaction tubes；
（4）Analyze RPA amplified productions；
The RPA methods are not for the purpose of disease detection and/or treatment.
7. RPA methods according to claim 6, which is characterized in that the RPA amplification reaction systems are calculated as with 50 μ l：
2.0 μ l of template DNA
2.0 μ l of sense primer
2.0 μ l of downstream primer
29.5 μ l of rehydration buffer
2.5 μ l of magnesium acetate
12 μ l of nuclease free pure water
Wherein, sense primer and the initial concentration of downstream primer are 0.25 μM, and the initial concentration of the magnesium acetate is 280mM.
8. RPA methods according to claim 6, which is characterized in that step（4）It is middle analysis RPA amplified productions method be：
RPA amplified productions are diluted, then the RPA amplified productions after dilution are examined with colloidal gold lateral flow immunochromatography test strips
It surveys.
9. RPA methods according to claim 8, which is characterized in that in the colloidal gold 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, which is characterized in that in the colloidal gold lateral flow immunochromatography test strips
Equipped with anti-fluorescein isothiocynate antibody detection line and biotinylated antibody nature controlling line；If colloidal gold lateral flow immunochromatography test paper
There is band in the anti-fluorescein isothiocynate antibody detection line of item, and biotinylated antibody nature controlling line is normal, then shows the sample
Show to be free of in the sample if being only band occur on biotinylated antibody nature controlling line containing 4 type of aviadenovirus serum in product
4 type of aviadenovirus serum.
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| CN109321677B (en) * | 2018-09-11 | 2022-05-03 | 迈克生物股份有限公司 | Method for specifically detecting H1 subtype A by combining RPA with immunofluorescence chromatography |
| CN109504800A (en) * | 2018-11-22 | 2019-03-22 | 李越希 | A kind of RPA method for detecting universal adenovirus hominis, its primer special and probe and purposes |
| CN109811069B (en) * | 2019-04-11 | 2020-11-24 | 中国农业大学 | Kit for rapidly detecting donkey-derived components in food and application thereof |
| CN110257562A (en) * | 2019-07-31 | 2019-09-20 | 河北农业大学 | A kind of the primer and probe combination and its application of RAA-LFD detection avian infectious laryngotracheitis virus |
| CN110938711A (en) * | 2019-12-20 | 2020-03-31 | 河北农业大学 | Real-time fluorescent RAA primer, probe and kit for detecting avian infectious laryngotracheitis virus and using method of real-time fluorescent RAA primer, probe and kit |
| CN111118212B (en) * | 2020-01-07 | 2023-11-14 | 河北农业大学 | Primer and probe combination for detecting newcastle disease virus, kit and detection method |
| CN111257555A (en) * | 2020-04-03 | 2020-06-09 | 安徽省疾病预防控制中心（省健康教育所） | Rapid detection method and test strip for lateral chromatography colloidal gold of new coronavirus nucleic acid recombinase mediated isothermal amplification |
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