CN108315491A - RPA- Sidestream chromatographies detection primer, probe and the kit of 3 type of bovine parainfluenza virus - Google Patents
RPA- Sidestream chromatographies detection primer, probe and the kit of 3 type of bovine parainfluenza virus 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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- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses primer, probe, kit and detection methods that a kind of RPA Sidestream chromatographies detect BPIV3 aerosols, include the primed probe liquid for the RPA primer and probes composition for detecting BPIV3 in the kit, wherein forward primer sequence such as SEQ ID No.1, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.Primed probe group RPA expanding effects used in the present invention are good, band high specificity, with other viral no cross reactions, have strong positive reaction in detection zone, increase the sensitivity of detection.System of the present invention uses the method that RPA Sidestream chromatography technologies establish quickly detection ox BPIV3 for the first time, and by specificity and sensitivity evaluation, can be used for clinical sites detection, and a kind of sensitive, reliable new method is provided for the Site Detection of BPIV3.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of application recombinase polymeric enzymatic amplification technology
(Recombinase Ploymerase Amplification, RPA) and combine Sidestream chromatography technology (Lateral Flow
Assay primer, probe and its kit of BPIV3) are detected.
Background technology
Ox parainfluenza is also known as transporting hot, is by 3 type of bovine parainfluenza virus (Bovine parainfluenza
Virus type 3, BPIV3) a kind of acute contact respiratory infectious disease caused by virus, weather cataclysm is taken place mostly in, it is long
The intensive vaccary of way transport cows and stocking density greatly.BPIV3 is also more important bovine respiratory syndrome
One of (Bovine respiratory disease complex, BRDC) cause of disease, mainly encroaches on respiratory tract, causes immunity of organism
Function and macrophage function decline, and cause infection animal immunosupress, and then secondary bacterial or mycoplasma infection, occur typical
BRDC, lead to serious bronchitis, capillary bronchitis and pneumonia.
Due to cultivated animals highly dense, a large amount of pathogenic microorganism is drained in infected animal exhalation, causes to give up interior high concentration
The accumulation of microbial aerosol, and by the exchange of gas inside and outside house, pathogenic microorganism is caused with atmospheric propagation, diffusion
Environmental organism pollutes and infectious disease transmission.BPIV3 can form aerosol in air, and virus can remote with the wind, fast propagation
Degree.It therefore, will be for the group of bovine parainfluenza by establishing a kind of detection method suitable for BPIV3 aerosols and clinical sample
Monitoring and forecasting and warning provide supporting technology.The diagnostic method of the disease mainly has pathogen separation, PCR method, ELISA experiments at present
With serum neutralization test etc., since these methods need accurate instrument and cumbersome test procedure, it is difficult to meet non-experiment
The requirement of Site Detection under room environmental.It is Site Detection, disease thus there is an urgent need for establishing a set of diagnostic method quickly, accurate, easy
Former environmental monitoring and epidemic situation forecasting and warning etc. provide the detection technique and means of a new generation.
Recombinase polymeric enzymatic amplification (recombinase ploymerase amplification, RPA) technology is different
In the nucleic acid detection technique of PCR, RPA is the reproduction process of the DNA in recombinase-mediated Imitating body.Recombinase is combined with primer
After forming Protein-DNA mixtures, double-stranded DNA homologous sequence is searched out, under the action of single-stranded DNA binding protein, template DNA
Unwinding, primer are matched with template DNA, and are replicated, expanded under the action of strand displacement archaeal dna polymerase, and single DNA template molecule obtains
To about 1012Amplified production.It is different from PCR, does not need annealing reaction process, can be under 37 DEG C~42 DEG C isothermys, instead
More than 20 min are answered to can be obtained amplified production (Forget et al., 2012).RPA can be with lateral flow immunochromatography technique phase
In conjunction with, in RPA amplification systems, the probe for needing the reverse primer of biotin labeling and fluorophor to mark, away from fluorescent base
There are one abasic sites (dSpacer), the site can be identified, cut by nfo ribozymes at 30 bases of group, generates new hydroxyl
End, and extend under archaeal dna polymerase effect, the RPA products with biotin and fluorophor double labelling are formed, chromatography is passed through
Membrane diffusion is captured by biotin ligand and fluorophor labelled antibody, is formed and has coloured detection line.This method detection time
It is short, 5min or so energy visual observations, naked eyes interpretation.
System of the present invention uses the method that RPA technologies establish quickly detection BPIV3 for the first time, and is commented by specificity and sensitivity
Valence can be used for clinical sites detection, and a kind of sensitive, reliable new method is provided for the Site Detection of BPIV3.
Invention content
To solve the above-mentioned problems, the present invention provides primer, probe groups that a kind of RPA- Sidestream chromatographies detect BPIV3
It closes, a kind of kit of detection BPIV3, which uses accurate, quick, easy.The present invention also provides a kind of application RPA skills
Art and the method for combining Sidestream chromatography technology detection BPIV3.
To achieve the above object, the present invention uses following technical proposals：
A kind of RPA- Sidestream chromatographies detect BPIV3 primer and probes, forward primer sequence as shown in SEQ ID No.1,
Reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
Forward primer BPIV3-F：5 '-TTTATCAATGAATTAGCAAACAAGAGAGAT-3 ', (SEQ ID No.1)；
Reverse primer BPIV3-R：
5 '-[Biotin] CCGATTTGTAATACTTGATAAGACTTCCCT-3 ', (SEQ ID No.2)；
Probe sequence BPIV3-P：5’-[FAM]GAGATGTACCTCTGGCAATCCATCCCTGACAAG[dSpacer]
AACCCAAAGATAAGAC[C3-spacer]-3’；(SEQ ID No.3).
It should be noted that：Different from routine PCR reaction, the length of primer is usually 30-35bp needed for RPA reactions, is visited
The length of needle sequence is 46-52bp, when design of primers in order to avoid formed inside primer and between secondary structure, length
Increase the increase for also making design of primers and selecting difficulty, therefore, the design and selection of primer are most important to the result of RPA.RPA
Technology is in starting conceptual phase, there is no special primer, probe design software, is also that its primer is set without a large amount of data
It counts principle and foundation is provided.Therefore, primer and probe combination of the invention be need from target sequence both ends design multipair primer into
Row optimization, screening can just obtain.
The present invention protects above-mentioned primer and probe to combine the application in the detection reagent for preparing BPIV3 accordingly.
Include for above by RPA skills in the kit the present invention also provides a kind of kit of detection BPIV3
Art detects the primer and probe combination of BPIV3.
The group of the primed probe liquid becomes：Forward primer and reverse primer final concentration are 0.4 μm of ol/L, detection probe
Final concentration of 0.12 μm of ol/L, amplification BPIV3 nucleotide sequence fragments be SEQ ID NO.4.
Further, further include in mentioned reagent box：Standard positive plasmid, buffer solution, eight union of enzymatic amplification, sterile double steamings
Water, reaction driving liquid, product dilution and Sidestream chromatography detection reagent.
The standard positive plasmid is T3- BPIV3-HN examines corresponding reaction for the positive control of BPIV3 detections
System and reaction condition can normal reaction, by the nucleotide sequence fragment SEQ of SEQ ID NO.5 and SEQ ID NO.6 amplifications
ID NO.7 and pEASY-T3The recombinant plasmid that carrier obtains after being connected..
The RPA Sidestream chromatographies detection reagent includes Sidestream chromatography test strips, hybridization check buffer solution (1 × PBS+0.1%
Tween20)。
The aseptic double-distilled water can be used as negative control or supply reaction system use, and preparation method is distilled water through high pressure
After sterilizing, it is dispensed into the tubule of sterilizing.
The buffer solution, eight union of enzymatic amplification, reaction driving liquid are commercially available in market.
Specifically, a kind of kit of detection BPIV3,29.5 μ L of buffer solution, BPIV3 are contained in every 50 μ L amplification systems
4.6 μ L of primed probe liquid, 12.4 μ L of aseptic double-distilled water, sample to be tested cDNA using positive criteria plasmid as positive control are respectively
1 μ L drive 2.5 μ L of liquid by 1 μ L of blank control, reaction of aseptic double-distilled water.
The present invention also provides a kind of methods detecting BPIV3 using RPA technologies and combination Sidestream chromatography technology, and step is such as
Under：
(1) total serum IgE for extracting sample to be tested carries out reverse transcription with reference to reverse transcription reagent box specification, obtains c DNA；
(2) it is designed for the primer and probe of BPIV3 detections, using cDNA as template, carries out RPA amplifications, amplification condition is：
After 38 DEG C of water-bath 4min, mixing, then 38 DEG C of water-bath 20min；Forward primer sequence is as shown in SEQ ID No.1 in the primer,
Reverse primer sequences are as shown in SEQ ID No.2, and the probe sequence is as shown in SEQ ID No.3.The spy synthesized under this condition
Needle expanding effect is best, and band specificity is good.
Preferably, RPA amplified productions are separately recovered through 1% agarose gel electrophoresis, are cloned into pEAST- in step (2)
T3 carriers connect, conversion, and blue hickie screening, picking white colony carries out bacterium colony PCR verifications.Positive restructuring bacterium is trained in LB
It supports overnight, kit extracts Plasmid DNA, and plasmid order-checking will be sequenced correct recombinant bacterium and shake bacterium culture, extracts Plasmid DNA, obtain
Positive plasmid is named as T3-BPIV3-HN.
(3) Sidestream chromatography test strips are applied to carry out amplified production detection.
Preferably, when two brown bands occur in test strips, one is located in quality control region, and one is located at detection zone, then ties
Fruit is the positive, shows to contain BPIV3 nucleic acid in sample；When test strips only have quality control region a brown band occur, detection zone does not have
There is band, then the result is that it is negative, show not containing BPIV3 nucleic acid in sample.
Beneficial effects of the present invention：
(1) present invention system uses the method that RPA- Sidestream chromatography technologies establish quickly detection ox BPIV3 for the first time, and passes through spy
Anisotropic and sensitivity evaluation can be used for clinical sites detection, and a kind of sensitive, reliable new side is provided for the Site Detection of BPIV3
Method.
(2) primer and probe combination using the present invention, detects BPIV3 clinical samples by RPA technologies, is had
There are higher sensitivity, specificity and repeatability.
The BPIV3HN gene primers that the present invention selects are to screen to obtain through many experiments, and specificity is good, other with ox
Virus includes bovine enteroviruses, ox stream fever virus, bovine viral diarrhea virus, vesicular stomatitis virus, ox infectious rhinotracheitis
The virus no cross reaction such as scorching virus.
Primed probe expanding effect used in the present invention is good, band high specificity, can be formed in detection zone higher
The primer-probe heterodimer of concentration, thus make test strips that strong positive reaction be presented, increase the sensitivity of detection, institute of the present invention
Establish the BPIV3 particles of detectable 3 copies of detection method.
(2) RPA technologies of the invention and the method for combining Sidestream chromatography technology detection BPIV3 aerosols, both have molecule
Biological Detection it is highly sensitive, high-throughput, and have the advantages that the specificity of immunology detection is good, easy to operate, be further without multiple
Miscellaneous instrument, particularly suitable for laboratories and the detection of the BPIV3 rapid screenings at scene.
(3) detection speed is fast：Compared with Standard PCR, it is not necessary to by denaturation, annealing, extend three steps, RPA reactions
Optimum temperature is between 37 DEG C -42 DEG C, and without denaturation, reaction can be completed in 20min or so at normal temperatures.
(4) complicated instrument and equipment is not needed, Site Detection is suitable for.The established detection method of the present invention can be in room temperature
Amplification, test strips Visual retrieval under isothermy, should not PCR instrument, fluorescence quantitative PCR instrument, electrophoresis apparatus, electrophoresis tank etc. it is complicated
Instrument and equipment, and RPA is not required to complicated sample treatment, can really realize portable live Rapid nucleic acid detection.
Description of the drawings
The RPA primers of Figure 1B PIV3, the agarose gel electrophoresis figure of probe groups；
Wherein 1 is F1/P1/R1 primed probe groups, and 2 be F1/P1/R2 primed probe groups, and 3 be F2/P1/R1 primed probes
Group, 4 be F2/P1/R2 primed probe groups, and 5 be F3/P1/R1 primed probe groups, and 6 be F3/P1/R2 primed probe groups, and PC is the positive
Control, NC are negative control, and M is DNA Marker DL2000；
The RPA primers of Fig. 2 BPIV3, the Sidestream chromatography test strips the selection result figure of probe groups；
Wherein 1 is F1/P1/R1 primed probe groups, and 2 be F1/P1/R2 primed probe groups, and 3 be F2/P1/R1 primed probes
Group, 4 be F2/P1/R2 primed probe groups, and 5 be F3/P1/R1 primed probe groups, and 6 be F3/P1/R2 primed probe groups, and PC is the positive
Control, NC is negative control；
The selection result figure of the RPA reaction temperatures of Fig. 3 BPIV3；
The selection result figure in the RPA reaction time of Fig. 4 BPIV3；
The agarose gel electrophoresis testing result figure of Fig. 5 RPA sensitivitys；
The Sidestream chromatography ELISA test strip result figure of Fig. 6 RPA sensitivitys；
The specific test result figure of Fig. 7 RPA Sidestream chromatography ELISA test strip methods；
Cause of disease is respectively bovine viral diarrhea virus (BVDV), infectious bovine rhinotrachetis virus (IBRV), ox stream pyreticosis
Malicious (BEFV), bovine coronavirus (BCoV), vesicular stomatitis virus (VSV), Bovine Respiratory Syncytial are viral (BRSV).
The RPA Sidestream chromatography ELISA test strip result figures of Fig. 8 partial clinical samples.
Specific implementation mode
With reference to embodiment, the present invention is further illustrated, it should which explanation, following the description is merely to solution
The present invention is released, its content is not defined.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
TwistAmp nfo Kits are purchased from TwistDX companies, Genline Hybridetect-1lateral flow
Strips is purchased from Milenia GmbH (Germany), reverse transcription reagent box (Code No.：D2639A) it is purchased from precious bioengineering
The DNA/RNA extractions of (Dalian) Co., Ltd, clinical sample use Punch-itTMNA-Sample Kit(Nanohelix,
Daejeon,South Korea)；Detection primer and probe are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.In embodiment not
The experimental method of actual conditions is indicated, usually according to conventional condition, such as Sambrook etc.《Molecular cloning：Laboratory hand
Volume》(New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or according to instrument or
Condition proposed by reagent manufacturer is operated.
One, the preparation of positive criteria plasmid
1. the design and synthesis of primer
By carrying out sequence alignment to BPIV3HN genes in GeneBank, determines conservative region, design 1 couple of primer HN-F：
5 '-GCAACCACAACAAAATAGC-3 ', HN-R：5 '-CGGAGTTGAGCACAACTG-3 ', the respectively SEQ in sequence table
ID NO.5 and SEQ ID NO.6 amplify PCR fragment (the SEQ ID NO.7 in sequence table), for positive criteria plasmid
Structure；All primers are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
The preparation of 2.BPIV3cDNA
With reference to viral DNA/RNA extracts kit specifications, extract BPIV3RNA, with reference to reverse transcription reagent box specification into
Row reverse transcription obtains cDNA.
3. the preparation of positive plasmid
CDNA prepared by method described above is template, carries out PCR amplification, and reaction system is 50 μ L：10×PCR Buffer
II(Mg2+Plus) 5 μ L, 8 μ L of 2.5mM dNTP Mixture, 0.5 μ L of LA Taq (5U/ μ L), 2 μ L of cDNA templates add respectively
Enter each 2 μ L of primer BPIV3-F and BPIV3-R of 10 μm of ol/L, sterilizing ultra-pure water adds to 50 μ L.Response procedures are 94 DEG C of pre-degenerations
3min, then 94 DEG C of 20s, 55 DEG C of 40s, 72 DEG C of 2min, 35 cycles；72 DEG C of extension 5min, 4 DEG C of preservations.
The pcr amplification product of BPIV3HN is that 641bp is separately recovered through 1% agarose gel electrophoresis, is cloned into pEAST-
T3Carrier connects, conversion, and blue hickie screening, picking white colony carries out bacterium colony PCR verifications.Positive restructuring bacterium is trained in LB
It supports overnight, kit extracts Plasmid DNA, and plasmid order-checking will be sequenced correct recombinant bacterium and shake bacterium culture, extracts Plasmid DNA, obtain
Positive plasmid is named as T3-BPIV3-HN.Two, the optimization and foundation of BPIV3RPA Sidestream chromatographies ELISA test strip method
The design of 1.RPA primers and probe
The key of RPA amplifications is the design of amplimer and probe.Related RPA primer and probes design related data
It is few, it there is no the principle of the software or maturation for design at present.PCR primer is not particularly suited for RPA, and RPA primers draw than general PCR
Object is about 30-35bp.Primer is too short to reduce recombination fraction, influence amplification rate and detection sensitivity；And primer length increases appearance
Easily formed inside primer and between secondary structure, increases of length also make the increase of design of primers and selection difficulty.Therefore,
Primer for RPA amplifications needs to be screened and optimized by a large amount of experiment.
The design of primer, probe refers to following factor under normal circumstances：(1) G/C content is 40%~60%；(2) as possible
Avoid occurring secondary structure inside primer；(3) primer is avoided to duplicate sequence.
General 46~52 bases of length of RPA probes avoid, formation heterodimer Chong Die with primer as possible.Probe 5 '
Hold flag F AM groups, intermediate THF substitutes G or C (dSpacer), and the both sides dSpacer avoid G, C as possible, dSpacer modifications with
5 ' end at least 30 bases, with 3 ' end at least 15 bases, 3 ' end C3- spacer is modified.
The present embodiment is directed to the conserved regions design of BPIV3HN genes a series of RPA primers and probe, is specifically shown in Table 1.
Table 1 is directed to the RPA primers and probe of BPIV3HN gene conserved regions designs
2. screening primer and probe using RPA amplified reactions
With 107The T3-BPIV3-HN of copy is template, and RPA methods expand the HN genes of BPIV3.Specific steps：
(1) primed probe group is separately added into centrifuge tube tube bottom：Wherein each 2.1 μ L of upstream and downstream primer (10 μm of ol/L),
0.6 μ L of LF probes (10 μm of ol/L), 29.5 1 μ L of μ L, T3-BPIV3-HN of buffer solution, use ddH2O supplies 47.5 μ L of volume, blows
Beat mixing；Each pair of primed probe group sets up negative control.
(2) 47.5 μ L buffer solutions are transferred in eight reaction tubes of 0.2mL TwistAmp nfo containing freeze-drying enzyme powder,
It is blown and beaten repeatedly through pipettor until being completely dissolved.
(3) magnesium acetate (280mmol/L) solution of 2.5 μ L is added into each reaction tube liquid, is reacted at once after mixing
Occur.
(4) reaction tube is put into 38 DEG C of thermostat water bath, handles 4min.
(5) after reacting 4min, reaction tube is taken out, continues to be put into 38 DEG C of thermostat water bath again after mixing to react
20min。
(6) detection pipe is taken, the product dilution that amplified production 1 μ L and 49 μ L are added wherein is put into inspection after mixing
Paper slip is tested, after 3~5min, the colour developing for passing through test strips carries out interpretation.
3. the selection result of primer and probe
BPIV3 primed probes group shown in table 1 is detected after RPA is expanded by agarose gel electrophoresis, primed probe group
6：The RPA expanding effects of F3/P1/R2 are best, and band specificity is good, without non-specific amplification；And other probe primers are to going out
Existing non-specific amplification, without purpose band, probe and downstream primer amplification efficiency are low the problems such as by ELISA test strip (figure
1)；For RPA amplified productions through Sidestream chromatography ELISA test strip, there is band, primed probe group 6 in quality control region：The RPA of F3/P1/R2
Product is most deep in the color of detection zone, shows that primed probe group 6 forms the primer-probe heterodimer of higher concentration, thus
Make test strips that strong positive reaction be presented；And although other primed probe groups also positive band can occur in the detection zone of test strips,
But it is shallower to detect color, shows to be formed by primer-probe heterodimer efficiency low, and has in conjunction with agarose gel electrophoresis data
Non-specific amplification, thus these primed probes are to may not apply to the detection of RPA.Therefore, primed probe group 6 is selected：F3/
P1/R2 carries out subsequent RPA reaction condition optimizations, specificity and sensitivity tests, and primed probe is respectively designated as
BPIV3-F、BPIV3-R、BPIV3-P。
The optimization of 4.RPA amplification reaction systems
Shown in table 2, the optimization of BPIV3-F, BPIV3-R, BPIV3-P usage amount is carried out, optimum results show upstream and downstream
Primer is respectively 2 μ L, probe is 0.6 μ L, and effect is preferable.In addition, the usage amount also directed to magnesium acetate is provided with 7 concentration gradients.
The amount that a concentration of 280mM/ μ L magnesium acetates are added in RPA reaction systems is respectively 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 2.5 μ L and 3
μ L, the results show that magnesium acetate dosage, which crosses major general, leads to the failure of amplification, 1 μ L and the above magnesium acetate are added in reaction system can be with
Obtained amplified production, and product increases with the increase of acetic acid magnesium amount, but add the magnesium acetate difference of 2.5 μ L and 3 μ L
Unobvious.2.5 μ L magnesium acetates are added in final determine in reaction system.
RPA reaction systems after optimized are：Each 2.0 μ L of upstream and downstream primer (10 μm of ol/L), 0.6 μ L of probe (10 μm of ol/
L), 1 μ L of 29.5 μ L of buffer solution, template cDNA, use ddH2Above-mentioned mixed liquor is added to containing freeze-drying enzyme powder by 12.4 μ L of O
In eight reaction tubes of 0.2mL TwistAmp nfo, blown and beaten repeatedly with pipettor until being completely dissolved.It is eventually adding the vinegar of 2.5 μ L
Sour magnesium (280mmol/L) starts reaction.38 DEG C of 4min, after mixing, then 38 DEG C of 20min.
In addition, also optimized to RPA reaction time, reaction temperature, use respectively 30 DEG C, 34 DEG C, 38 DEG C, 42 DEG C, 45
DEG C and 48 DEG C carry out RPA reaction temperatures optimize, the RPA amplification efficiencies highest (Fig. 3) of 38 DEG C of reaction temperature；It passes through respectively
5min, 10min, 15min, 20min, 25min, 30min progress RPA reaction time optimize, reaction time 20min or more
Time can reach preferable amplification efficiency, and the RPA reaction time of optimization is 20min (Fig. 4).
Table 2RPA reacts primer and probe usage amount Combinatorial Optimization table
Three, the sensitivity of BPIV3RPA Sidestream chromatographies ELISA test strip method, repeatability, specific detection
The sensitivity analysis and repeatability detection of 1.RPA Sidestream chromatography ELISA test strip methods
By standard positive plasmid T3- BPIV3-HN carries out 10 times through PBS and is serially diluted 5 × 105~5 copies and 1 copy,
Using the RNA, reverse transcription cDNA of kit extraction virus；RPA amplifications are carried out with the RPA conditions of optimization, reaction system is：Up and down
Each 2.0 μ L of primer (10 μm of ol/L) are swum, 0.6 μ L of probe (10 μm of ol/L), buffer solution 1 μ L of 29.5 μ L, template cDNA use ddH2O
Above-mentioned mixed liquor is added in eight reaction tubes of 0.2mL TwistAmp nfo containing freeze-drying enzyme powder, uses liquid relief by 12.4 μ L
Device is blown and beaten repeatedly until being completely dissolved.The magnesium acetate (280mmol/L) for being eventually adding 2.5 μ L starts reaction.38 DEG C of 4min, mixing
Afterwards, then 38 DEG C of 20min.
The template concentrations of above-mentioned each gradient do 3 repetitions, by RPA Sidestream chromatography ELISA test strips, verify RPA effluents
Batch interior repeatability of chromatograph test strip technology；In addition, being repeated once at interval of 2d, 3 repetitions are carried out altogether, verify RPA effluent layers
Analyse Lateral Flow Strip batch between repeatability.The RPA products obtained carry out Sidestream chromatography ELISA test strip respectively.The result shows that
The detection of RPA-LFD is limited to 5 copies (Fig. 5).
The specificity analysis of 2.RPA Sidestream chromatography ELISA test strip methods
With reference to viral DNA/RNA extracts kit specifications, the DNA of infectious bovine rhinotrachetis virus (IBRV) is extracted,
Extract bovine viral diarrhea virus (BVDV), ox stream fever virus (BEFV), bovine coronavirus (BCoV), vesicular stomatitis virus
(VSV), the RNA of Bovine Respiratory Syncytial viral (BRSV) carries out reverse transcription with reference to reverse transcription reagent box specification, obtains
cDNA。
Establish RPA methods according to above-mentioned steps and carry out specific test, respectively with the DNA of IBRV, BVDV, BEFV, BCoV,
The cDNA of VSV, BRSV are template, with ddH2O is negative control, carries out RPA amplifications, and experiment is repeated 3 times.
All RPA products carry out Sidestream chromatography ELISA test strip, as shown in fig. 7, it can be seen from the figure that RPA is expanded
BPIV3 specificity is good, with the viral no cross reaction of other detections.Therefore, primed probe group BPIV3-F, BPIV3-R, BPIV3-
P can be used for establishing the RPA Sidestream chromatography test strips methods of detection BPIV3, be suitable for identifying the detection of unknown sample BPIV3.
Four, the assembling and sensitivity of RPA Sidestream chromatographies detection kit, repetitive test
Each 200 μ L of primer BPIV3-F, BPIV3-R, 60 μ L of probe BPIV3-P are mixed, 50 μ L/ pipes of packing, standard male
Property grain T3- BPIV3-HN is dispensed, 50 μ L/ pipes of packing, as positive control；Then with buffer solution, eight union of enzymatic amplification, sterile
Distilled water, reaction driving liquid, sidestream immune chromatograph test strip, product dilution are assembled into RPA Sidestream chromatography detection kits.
Assembled RPA Sidestream chromatography detection kits are utilized, by T in embodiment 33The 5 of-BPIV3-HN carriers copy into
Line sensitivity detects, and reaction system is：4.6 μ L of primed probe liquid, buffer solution 29.5 μ L, template cDNA 1 μ L, ddH2O 12.4μ
L, above-mentioned mixed liquor is added in eight union of enzymatic amplification, is blown and beaten repeatedly with pipettor until being completely dissolved.It is eventually adding 2.5 μ L
Reaction driving liquid start reaction.38 DEG C of 4min, after mixing, 38 DEG C of 20min.Three repetitions are done, the RPA amplified productions of 1 μ L are taken
It is diluted with the product dilution of 49 μ L, sidestream immune chromatograph test strip is detected.The result shows that the RPA effluent layers assembled
The T3-BPIV3-HN for 5 copies that detection kit can detect is analysed, experiment is repeated 3 times.
Five, the detection of clinical sample
1. the acquisition of clinical sample
Aerosol sample：The different zones (cow house, sports ground, milking parlour etc.) for selecting cattle farm, it is empty using MS-I types
Gas microbe sampling box passes through the Porton samplers sample air samples of multifunctional microbial sampler (Qingdao, MS-1 types).
Tripod is installed first, and sampler placing height is then 10mL sterile phosphate buffers (PBS) to be injected away from ground 1.5m
Proton samplers, while a drop olive oil is added dropwise, it is put into three foot fixing brackets fixed.By Porton impact type flow straighteners
One end is connected on host air intake, and the other end is connected to rubber tube on the gas outlet of Proton samplers.Sampling flow be 5~
35L/min, sampling time 30min.The collection liquid in sampler the preservation of 15mL centrifuge tubes is collected into after sampling to be used for
Detection.
Nose swab sample：The nose swab sample with respiratory tract clinical symptoms ox is acquired, is put in containing 5mL PBS's
It is preserved in centrifuge tube for detecting.
2. utilizing the nucleic acid of live extracts kit Punch-itTM NA-Sample Kit rapid extraction clinical samples
(1) in the centrifuge tube of 1.5mL, the lysate of 50 μ L is added, adds the sample of 10 μ L, uniformly mix；
(2) above-mentioned lysate is added in the sample aperture of kit, until solution is absorbed by filter membrane completely；
(3) the cleaning solution 3-5min of 200 μ L is added on filter membrane；
(4) the filter membrane progress that 1mm sizes are removed from sample aperture filter membrane is to be checked.
3. reverse transcription prepares cDNA
Reverse transcription, 5 × RT buffer, 2 μ L, 1 μ L of random primer, AMV reversions are carried out with reference to reverse transcription reagent box specification
Record 1 μ L of enzyme, ddH25 μ L of O, the 1mm size filter membranes containing RNA.30 DEG C of 10min, 42 DEG C of 30min, 95 DEG C of 5min, by reaction tube
It is placed on ice.
4. the detection of clinical sample
Sample to be tested is 13 parts of aerosol sample, the nose swab sample 21 through this laboratory using the detection of fluorescent quantitation method
Part and 15 parts of negative samples.Utilize live extracts kit Punch-itTMNA-Sample Kit rapid extraction clinical samples
Total serum IgE, reverse transcription cDNA.CDNA with 49 parts of samples of extraction is respectively template, using positive plasmid as positive control, ddH2O
For negative control, the detection of BPIV3 is carried out using the RPA Sidestream chromatography detection kits that embodiment 4 assembles.Reaction system is such as
Under：4.6 μ L of primed probe liquid, buffer solution 29.5 μ L, template cDNA 1 μ L, ddH212.4 μ L of O, above-mentioned mixed liquor is added to
In eight union of enzymatic amplification, blown and beaten repeatedly with pipettor until being completely dissolved.The reaction driving liquid for being eventually adding 2.5 μ L starts instead
It answers.38 DEG C of 4min, after mixing, 38 DEG C of 20min.The RPA amplified productions of 1 μ L are taken to be diluted with the product dilution of 49 μ L, effluent is exempted from
Epidemic disease chromatograph test strip is detected.If positive detection band occurs in the detection zone of test strips, contain BPIV3 in sample to be tested；
If the detection zone of test strips does not occur positive detection band, there is no BPIV3 pathogen in sample to be tested.
It is detected through RPA Sidestream chromatography detection kits, is in 34 parts of the sample of the BPIV3 positives, 15 parts of the sample being negative (figure
8), this result is compareed with SYBR Green I fluorescent quantitation PCR testing results, as a result completely the same.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Normal University
<120>RPA- Sidestream chromatographies detection primer, probe and the kit of 3 type of bovine parainfluenza virus
<130> 2010
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence
<400> 1
tttatcaatg aattagcaaa caagagagat 30
<210> 2
<211> 30
<212> DNA
<213>Artificial sequence
<400> 2
ccgatttgta atacttgata agacttccct 30
<210> 3
<211> 50
<212> DNA
<213>Artificial sequence
<400> 3
gagatgtacc tctggcaatc catccctgac aagtaaccca aagataagac 50
<210> 4
<211> 311
<212> DNA
<213>Artificial sequence
<400> 4
tttatcaatg aattagcaaa caagagagat caacaggagg taccaataca gagaatgact 60
catgacagcg ggattgaacc tctaaaccca gacaaattct ggagatgtac ctctggcaat 120
ccatccctga caagtaaccc aaagataaga ctaataccag gtcctagtct gttagcagca 180
tctaccactg taaatggttg tataaggatc ccgtccttcg taatcaacaa tctaatatat 240
gcttacactt ccaaccttat tgttcaaggt tgccaggata tagggaagtc ttatcaagta 300
ttacaaatcg g 311
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
gcaaccacaa caaaatagc 19
<210> 6
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<213>Artificial sequence
<400> 6
cggagttgag cacaactg 18
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<211> 641
<212> DNA
<213>Artificial sequence
<400> 7
gcaaccacaa caaaatagca tcacaacaaa tgagaaaaga attcgcagaa attgagaaaa 60
aaatccagca agctgcagat gaaattggta catcaataca atcgggaata aacacaagac 120
tcctaactat ccagagtcat gtccaaaatt atattccatt atctttgaca caacaaatgt 180
cggatcttag aaaatttatc aatgaattag caaacaagag agatcaacag gaggtaccaa 240
tacagagaat gactcatgac agcgggattg aacctctaaa cccagacaaa ttctggagat 300
gtacctctgg caatccatcc ctgacaagta acccaaagat aagactaata ccaggtccta 360
gtctgttagc agcatctacc actgtaaatg gttgtataag gatcccgtcc ttcgtaatca 420
acaatctaat atatgcttac acttccaacc ttattgttca aggttgccag gatataggga 480
agtcttatca agtattacaa atcgggatca tcactataaa ttcagacctg gtacctgact 540
taaaccctag agttacacat acttttaata ttgatgataa tagaaagtcc tgttctttag 600
cactactaaa tacagatgta tatcagttgt gctcaactcc g 641
Claims (10)
1. a kind of primer and probe combination of RPA- Sidestream chromatographies detection BPIV3 aerosols, which is characterized in that forward primer sequence
As shown in SEQ ID No.1, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.
2. primer and probe described in claim 1 combines the application in the detection reagent for preparing BPIV3.
3. a kind of kit of detection BPIV3, which is characterized in that the kit includes by primer described in claim 1 and spy
The primed probe liquid of needle composition.
4. kit as claimed in claim 3, which is characterized in that the group of the primed probe liquid becomes：Forward primer and anti-
It is 0.4 μm of ol/L, final concentration of 0.12 μm of ol/L of probe to primer final concentration, amplification BPIV3 nucleotide sequence fragments are
SEQ ID NO.4。
5. kit as claimed in claim 2, which is characterized in that the kit further includes：Standard positive plasmid, buffer solution,
Eight union of enzymatic amplification, aseptic double-distilled water, reaction driving liquid, product dilution and Sidestream chromatography test strips.
6. kit as claimed in claim 5, which is characterized in that the standard positive plasmid is T3- BPIV3-HN, by SEQ
Nucleotide sequence fragment SEQ the ID NO.7 and pEASY-T of ID NO.5 and SEQ ID NO.6 amplifications3Carrier obtains after being connected
Recombinant plasmid.
7. kit as claimed in claim 5, which is characterized in that contain 29.5 μ L of buffer solution, BPI in every 50 μ L amplification systems
Corresponding 4.6 μ L of primed probe liquid of V3,12.4 μ L of aseptic double-distilled water, sample to be tested cDNA are positive right with standard positive plasmid
According to respectively 1 μ L or using aseptic double-distilled water as 1 μ L of blank control, 2.5 μ L of reaction driving liquid.
8. a kind of method detecting BPIV3 using RPA technologies and combination Sidestream chromatography technology, which is characterized in that steps are as follows：
(1) total serum IgE of sample to be tested is extracted, reverse transcription obtains cDNA；
(2) it is designed for the primer and probe of BPIV3 detections, using cDNA as template, carries out RPA amplifications, amplification condition is：38℃
After water-bath 4min, mixing, then 38 DEG C of water-bath 20min；It is characterized in that, which is characterized in that forward primer sequence such as SEQ ID
Shown in No.1, reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3；
(3) Sidestream chromatography test strips are applied to carry out amplified production detection.
9. detection method as claimed in claim 8, which is characterized in that RPA amplified productions are solidifying through 1% agarose in step (2)
Gel electrophoresis is separately recovered, and is cloned into the connection of pEAST-T3 carriers, conversion, blue hickie screening, picking white colony, progress bacterium colony
PCR is verified；By positive restructuring bacterium, overnight incubation, kit extract Plasmid DNA in LB, and plasmid order-checking is correctly heavy by sequencing
Group bacterium shakes bacterium culture, extracts Plasmid DNA, obtains positive plasmid, is named as T3-BPIV3-HN.
10. detection method as claimed in claim 8, which is characterized in that expanded using Sidestream chromatography test strips in step (3)
Increase production analyte detection, when two brown bands occur in test strips, one is located in quality control region, and one is located at detection zone, then result is
The positive shows to contain BPIV3 nucleic acid in sample；When test strips only have quality control region a brown band occur, detection zone does not have item
Band, then the result is that it is negative, show not containing BPIV3 nucleic acid in sample.
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| CN113234860A (en) * | 2021-05-31 | 2021-08-10 | 中国农业科学院特产研究所 | Dual detection primer and probe combination for bovine viral diarrhea virus and bovine parainfluenza virus type 3 |
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