CN104862419B - A kind of primer, probe and kit for detecting infectious bovine rhinotrachetis virus - Google Patents

A kind of primer, probe and kit for detecting infectious bovine rhinotrachetis virus Download PDF

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CN104862419B
CN104862419B CN201510292511.XA CN201510292511A CN104862419B CN 104862419 B CN104862419 B CN 104862419B CN 201510292511 A CN201510292511 A CN 201510292511A CN 104862419 B CN104862419 B CN 104862419B
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primer
kit
probe
ibrv
rpa
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CN104862419A (en
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何洪彬
侯佩莉
王洪梅
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Dairy Cattle Research Center Shandong Academy of Agricultural Science
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Dairy Cattle Research Center Shandong Academy of Agricultural Science
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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

A kind of primer, probe and kit for detecting infectious bovine rhinotrachetis virus.It is used to combine by the primer and probe of RPA technology for detection infectious bovine rhinotrachetis viruses the invention discloses a kind of, its forward primer sequence is as shown in SEQ ID No.1, and reverse primer sequences are as shown in SEQ ID No.2, and probe sequence is as shown in SEQ ID No.3.The invention also discloses the kit detected for infectious bovine rhinotrachetis virus.Primer and probe using the present invention is detected, only need by carrying out viral DNA coarse extraction to clinical sample and carrying out RPA isothermal duplications, as a result can clearly be shown in Sidestream chromatography test strips, thermal cycle reaction is not required, it need not be expanded in PCR instrument, have the advantages that high sensitivity, high specificity, response procedures are simple, detection time is short, detect, be with a wide range of applications suitable for clinical sites under non-lab environment.

Description

A kind of primer, probe and kit for detecting infectious bovine rhinotrachetis virus
Technical field
The invention belongs to biological technical field, and in particular to one kind applies recombinase polymeric enzymatic amplification technology (Recombinase Ploymerase Amplification, RPA) and combine Sidestream chromatography technology (Lateral Flow Assay primer and probe combination and its kit used in infectious bovine rhinotrachetis virus) are detected.
Background technology
Infectious bovine rhinotrachetis virus (Infectious bovine rhinotracheitis virus, IBRV) is drawn The infectious bovine rhinotrachetis risen, is a kind of acute, hot, contagious disease of ox, special clinically in a variety of types of presentation It is not that the disease can cause immunosupress, the bacterium infection of Secondary cases can cause more serious breathing problem.This disease is alive at present It is widely current in the range of boundary, and the disease drastically influence international ox industry production trade, be arranged by World Organization for Animal Health (OIE) For B class transmissible diseases.China detects IBRV in late 1970s from the milk cow of New Zealand's import, due to not having very Good immune protection measure, has this virus infection in the cows of subsequent China's most laboratories, fattening, milk production to cows and numerous It is very big to grow influence, huge economic loss is caused to cattle farm.The diagnostic method sick to this mainly has pathogen separation, PCR side at present Method, ELISA experiments and serum neutralization test etc., it is difficult since these methods need accurate instrument and cumbersome test procedure To meet the requirement of Site Detection under non-lab environment.Thus there is an urgent need for establishing a set of quick, accurate, easy diagnostic method, The detection technique and means of a new generation are provided for clinical sites detection and epidemic monitoring etc..
Recombinase polymeric enzymatic amplification (Recombinase Polymerase Amplification, RPA), is a kind of different In the nucleic acid detection technique of PCR, mainly there are the recombinase with reference to single-chain nucleic acid, single-stranded DNA binding protein and strand displacement DNA polymerizations Three kinds of enzymes of enzyme participate in.Its principle is the Protein-DNA mixtures combined to form using recombinase and primer, can be sought in double-stranded DNA Look for homologous sequence.With the help of single-stranded DNA binding protein, template DNA is set to unwind, primer starts pairing with template DNA and formed 3 ' C-terminals needed for replicating, duplication extension is carried out under the action of archaeal dna polymerase, new DNA complementary strands are formed, to template On target area carry out exponential amplification.The design of primer sequence is with selecting the result to RPA most important.Expanded in RPA The probe of the reverse primer of one biotin labeling of addition and 5 ' fluorescent markers in system, 30 alkali of fluorophor of probe mark Ji Chu has an abasic site (dSpacer), which is the substrate that DNA repairs enzyme effect, can be identified and cut by ribozyme nfo Cut, produce 3 ' new C-terminals, duplication extension is carried out under the action of archaeal dna polymerase, so that double mark signals are produced with amplification The accumulated synchronized of thing, testing result can be combined in anti-FAM gold marks and antibiotin is captured and shown in the Sidestream chromatography test strips of antibody Show.
Research for RPA technologies at present is still in the starting stage, does not both at home and abroad also examine RPA technologies applied to IBRV The report of survey.Present invention system establishes the method for quick detection IBRV using RPA technologies first, and is commented by specificity and sensitivity Valency, is detected available for clinical sites, and a kind of sensitive, reliable new method is provided for the Site Detection of IBRV.
The content of the invention
It is applied to the blank of IBRV detection fields for RPA technologies, the present invention provides a kind of accurate, quick, easy inspection Survey the RPA detection methods of IBRV.Only need by carrying out viral DNA coarse extraction to clinical sample and carrying out RPA isothermal duplications, as a result Can clearly it be shown in Sidestream chromatography test strips, it is not necessary to thermal cycle reaction, it is not necessary to expanded, had sensitive in PCR instrument Height, high specificity, the advantages that response procedures are simple, detection time is short are spent, is detected suitable for clinical sites under non-lab environment, It is with a wide range of applications.
To achieve the above object, the present invention uses following technical proposals：
It is a kind of to be used to combine by the primer and probe of RPA technology for detection IBRV, its 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.
Forward primer：5’-TTCCGACCAGCGCCGAATCTTGGATGCAGTACT-3’；(SEQ ID No.1)
Reverse primer：5 '-GGCCAAAACCGCTTTCAGAAGCAGAGCAAGGTGA-3 ' (5 '-end is marked with Biotin)； (SEQ ID No.2)
Probe sequence：5’-CCGCAAAGTGCCGAGGGATGTCCTTGTAGT【dSpacer】CAGGTCCACCTTCCGCT- 3 ' (probe 5 '-end is marked with FAM, at the position of 30, distance 5 '-end base or so with dSpacer substitute a base, 3 ' end Blocked with C3-spacer).
It should be noted that：Different from Standard PCR reaction, the length of primer is usually 30-35bp needed for RPA reactions, is visited The length of pin sequence is 46-52bp, during design of primers in order to avoid formed inside primer and between secondary structure, its length Increase also makes design of primers and selects the increase of difficulty, and 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 set without substantial amounts of data for its primer Count 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.
Present invention also offers a kind of kit of detection IBRV, include in the kit for being examined by RPA technologies Survey the primer and probe combination of IBRV.
Further, further included in the kit：IBRV positive control templates, RPA amplifing reagents, deionized water and side Fluid layer analyses detection reagent.IBRV positive control templates, RPA amplifing reagents, deionized water are formed together with primer and probe combination Amplification system.
The RPA amplifing reagents include Rehydration buffer solutions, recA recombinases, the strand displacement of Escherichia coli Archaeal dna polymerase, single-stranded DNA binding protein (SSB), 280mM magnesium acetates (MgAc) solution.
The RPA Sidestream chromatographies detection reagent includes Sidestream chromatography test strips, hybridization check buffer solution (1 × PBS+0.1% Tween20)。
The preparation method of the IBRV positive control templates is：Respectively shown in SEQ ID No.1 and SEQ ID No.2 The upstream and downstream design primer of primer pair：
Sense primer:5’-GCCAAGCGCAGCAGGCAGGTGAAT-3’(SEQ ID No.4)；
Anti-sense primer:5’-CGACTGGGCGTACATCTCGGAGAA-3’(SEQ ID No.5)；
PCR amplification is carried out by template of the DNA of IBRV, reaction system is 50 μ L：10×LA 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 template, the upstream and downstream of 10 μm of ol/L Each 2 μ L of primer, sterilizing ultra-pure water add to 50 μ L.Response procedures are 94 DEG C of pre-degeneration 3min, subsequently into 94 DEG C of 30s, 55 DEG C 30s, 72 DEG C of 40s, carry out 31 circulations altogether；72 DEG C of extension 10min, finally stop at 4 DEG C.PCR product is through 1% Ago-Gel Electrophoresis, recycling purpose segment rear clone to pEAST-T3 carriers, (for conventional carrier, commercialization is bought) blue hickie screening weight Group plasmid, send Hua Da gene to be sequenced, sequencing result is compared, correct recombinant plasmid is IBRV positive control moulds Plate.
It is each containing forward primer, reverse primer in every 50 μ L amplification systems specifically, a kind of kit of detection IBRV 2.1 μ L (10 μm of ol/L), 0.6 μ L of probe (10 μm of ol/L), 2 μ L, Rehydration buffer solution 29.5 of IBRV positive control templates 2.5 μ L of μ L, 11.2 μ L of deionized water and magnesium acetate solution (280mM).
The present invention also provides a kind of using RPA technologies and as follows with reference to the method for Sidestream chromatography technology for detection IBRV, step：
(1) using the IBRV DNA of rapid extraction virus genom DNA kit extraction cell culture and virus supernatant；
(2) RPA specific primers and probe are designed according to IBRV conservative regions, to RPA amplification kit recommendation responses Forward primer, each 2.1 μ L of reverse primer (10 μm of ol/L), 0.6 μ L of probe (10 μm of ol/L), template are added in 50 μ L amplification systems 2 μ L of DNA, 29.5 μ LRehydration buffer solutions, 11.2 μ L deionized waters and 2.5 μ L magnesium acetate solutions (280mM), in containing In the 0.2mL TwistAmp nfo reaction tubes of lyophilized enzyme powder, reacted 25 minutes for 38 DEG C in thermostat water bath；
(3) 1 μ L amplified productions are taken, are detected using Sidestream chromatography test strips, if being shown in Sidestream chromatography test strips Band and control band are detected, then testing result is the positive, if test strips only show control band, result is feminine gender.
A set of primer and probe combinations that fast and effective can detect IBRV components can be filtered out using the above method.
Using known viruse titre as 8.0 × 107TCID50The IBRV of/mL with deionized water carry out 10 times be serially diluted after, carry Detection is synchronized after taking DNA, the results show can detect 0.8TCID50Target molecules, it was demonstrated that this method have it is higher Sensitivity.
With 8.0 × 103TCID50The DNA of IBRV samples carries out RPA as template using the primer and probe of above-mentioned optimization Isothermal duplication, amplified production shown in Sidestream chromatography test strips detection band and control band, and using other viral cDNA as Template amplification only shows control band, it was demonstrated that this method has preferable specificity.
With the 0.8TCID of extraction50The DNA of IBRV samples carries out RPA as template according to primer and probe described above Isothermal duplication, carries out 3 repetitions, amplified production is detected in Sidestream chromatography test strips respectively to the template.As a result phase is confirmed The amplified production of same amount template Sidestream chromatography test strips reaction zone show the identical detection band of brightness and control band, have compared with Good repeatability.
Beneficial effects of the present invention：
(1) primer and probe using the present invention combination, the method being detected by RPA technologies to IBRV, have compared with High sensitivity, specificity and repeatability.
(2) RPA technologies of the invention and the method for combining Sidestream chromatography technology for detection IBRV, can be not only used for clinical blood, The detection of the routine clinical sample such as milk sample, tissue, can also be detected the trace samples such as Virus Aerosol.The present invention need pair Clinical sample carries out viral genome coarse extraction, and special instrument is not required in constant-temperature amplification process, amplification stage, as a result can lead to Cross vision visual inspection, it is not necessary to which detector, and reaction speed is fast, sensitiveness is high, can be in a non-laboratory environment in the short time It can complete the clinical sites detection of IBRV.
Brief description of the drawings
The foundation of Fig. 1 IBRV diagnostic methods, wherein, 1：RPA kits provide primer, probe and template positive control；2： IBRV negative controls, template H2O；3：IBRV genomic DNAs.
Fig. 2 sensitivity techniques, template is followed successively by virus titer as 8.0 × 10 from 1 to 107-8.0×10-2TCID50IBRV samples The DNA of product.
The specific detection of Fig. 3 IBRV, wherein, 1：Infectious bovine rhinotrachetis virus (IBRV)；2：Bovine respiratory closes born of the same parents Precursor virus (BRSV)；3：Bovine viral diarrhea virus (BVDV)；4：Bovine parainfluenza type-3 virus (BPIV-3)；5：Bovine enteroviruses (BEV)；6：Bovine coronavirus (BcoV)；7：Bovine rota (BRV)；8：Bovine epizootic fever virus (BEFV).
The repeatability detection of Fig. 4 IBRV, template 0.8TCID50The DNA of IBRV samples.
Fig. 5 clinical samples detect, wherein, 1：Positive control；2：Negative control；3-16：IBRV positive samples；17-22： IBRV negative samples.
Embodiment
With reference to embodiment, the present invention is further illustrated, it should which explanation, the description below is merely to solution The present invention is released, its content is not defined.
Some materials and reagent used are as follows in example below：Molecular biology reagents, TwistAmp nfo Kits purchases From TwistDX companies；Genline Hybridetect-1lateral flow strips are purchased from Milenia GmbH (Germany)；LA Taq DNA Polymerase, it is limited that viral DNA/RNA extracts kits are purchased from precious bioengineering (Dalian) Company；RevertAidTMFirst Strand cDNA Synthesis Kit (catalog number (Cat.No.)s：K1622 it is) public purchased from Fermentas Department；PEASY-T3 Cloning Kit are purchased from Beijing Quanshijin Biotechnology Co., Ltd；Other biochemical reagents are import point Dress or domestic analysis are pure.Primer and probe is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
Instrument includes：Thermostat water bath, centrifuge, vortex instrument, spectrophotometer and pure water meter etc..
It is not specified the experimental method of actual conditions in embodiment, usually according to conventional condition, such as Sambrook etc. 《Molecular cloning：Laboratory manual》(New York:Cold Spring Harbor Laboratory Press, 2001) institute in Condition is stated, or is operated according to the condition proposed by instrument or reagent manufacturer.
Embodiment 1：The design and screening of primer and probe
The design effectively of primer and probe is the most critical link for determining Success in Experiment.However, RPA technologies are in starting Conceptual phase, there is no special primer, probe design software, also without substantial amounts of data for its design of primers principle provide according to According to.Need to optimize from the target sequence both ends multipair primer of design in experiment at present, screen, which requires pole It is stringent, and the replacement or increase and decrease of Individual base can all have an important influence on experimental result.Usually require to consider during design following Several factors：(1) primer length requirement be 30-35bp, and probe length requires to be 46-52bp, and G/C content is kept away in 40%-60% Exempt to occur secondary structure inside primer and avoid primer from duplicating sequence.(2) detection amplified fragments are less than 500bp.(3) avoid Dimer is formed between primer and probe, influences the accuracy of result.
Conserved sequence in this experiment according to the IBRV (accession number AJ004801.1) delivered in GenBank devises 4 Probe, 3 sense primers and 3 anti-sense primers have been separately designed in the both sides of every probe.With viral DNA rapid extraction reagent The genomic DNA of box extraction cell culture and virus is template, and the different primers, probe combinations to design carry out RPA amplification screenings, Negative control is made to primer, the probe amplification of design using deionized water as template at the same time, the mould provided with RPA amplification kits Positive control is done in plate, primer and probe combination, and finally selecting pair for amplification product can show clearly in Sidestream chromatography test strips Test strip and control stripes band (see Fig. 1), the primer and probe sequence filtered out see SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 (being shown in Table 1).
Table 1 screens obtained primer and probe sequence
Note：Probe 5 '-end is marked with FAM, and distance 5 ' holds and substitutes an alkali with dSpacer at the position of 30 bases or so Base, 3 ' ends are blocked with C3-spacer.
Biotin：Biotin；FAM：Luminophore；dSpacer：Abasic site；C3-spacer：Polymerase extension resistance Disconnected group.
Embodiment 2：The foundation and investigation of laboratory IBRV RPA amplification detection methods
1. the foundation of laboratory IBRV RPA amplification detection methods
The extraction of 1.1 IBRV genomic DNAs
200 μ L cell culture and virus supernatants are taken, IBRV is extracted using rapid extraction virus genom DNA kit DNA, is finally dissolved in 50 μ L deionized waters.
1.2 RPA are expanded
Using PRA methods amplification IBRV distinguished sequences in the present embodiment, concretely comprise the following steps：
(1) each 2.1 μ L of upstream and downstream primer (10 μm of ol/L), 0.6 μ L of LF probes that embodiment 1 designs are added into centrifuge tube (10 μm of ol/L), 29.5 μ L of Rehydration buffer solutions, 2 μ L of template DNA, use ddH2O supplies 47.5 μ L (table 2) of volume, Of short duration centrifugation after whirlpool mixes；
Table 2RPA amplification systems
(2) the 47.5 above-mentioned mixed liquors of μ L are transferred in the 0.2mL TwistAmp nfo reaction tubes containing lyophilized enzyme powder, Blown and beaten repeatedly until whole grain dissolution with pipettor；
(3) magnesium acetate solution of 2.5 μ L (280mmol/L) is added into each reaction tube, the whirlpool that firmly turns upside down mixes 8-10 times even, reaction occurs immediately；
(4) reaction tube is put into 38 DEG C of thermostat water bath, handles 4min；
(5) after reacting 4min, reaction tube is taken out, the whirlpool that firmly turns upside down mixes 8-10 times, and 38 are put into after of short duration centrifugation DEG C thermostat water bath in the reaction was continued 21min, obtain RPA reaction products.
1.3 Sidestream chromatography test strips carry out amplified production detection
Take a certain number of Sidestream chromatography test strips (dipstick, Milennia GenLine HybriDetect MGHD1), it is marked for different detection catalogue number(Cat.No.)s.Each detection sample takes 99 μ L hybridization check buffer solutions (HybriDetect Assay Buffer), adds in reaction tube.1 μ L hybridization reactions product (RPA reaction products) is taken to centrifuge Mixed in pipe, hybridization reaction solution and hybridization check buffer solution totally 100 μ L.The example reaction area of test strips is added into reaction solution, is incubated Educate 5 minutes, after incubation, test strips are taken out from reaction solution, are observed immediately.If test strips reaction zone show two it is visible Band, then detect sample as the positive；If test strips reaction zone control line shows that clearly, detection line has no band, then detects sample This is feminine gender.
The investigation of 2.IBRV RPA amplification detection methods
The sensitiveness of 2.1 IBRV RPA amplification detection methods is investigated
It is 8.0 × 10 by known viruse titre7TCID50It is dilute that the IBRV virus liquids of/mL with deionized water carry out 10 times of series After releasing, using the DNA of virus genom DNA rapid extraction kit extraction virus, it is template to draw 2 μ L IBRV DNA respectively, Expanded by " 1.2 primer amplification " operating method, take 1 μ L hybridization reactions product (RPA reaction products) in Sidestream chromatography test paper Shown on bar, the results showed that, it can detect 0.8TCID50Target molecules (Fig. 2), illustrate to be examined with the primer that designs of the present invention Surveying IBRV has higher sensitivity and accuracy, and easy to operate.
The specificity investigation of 2.2 IBRV RPA amplification detection methods
It is 2.0 × 10 to take 200 μ L bovine viral diarrhea virus (BVDV) titres6.0TCID50/ mL, bovine parainfluenza type-3 virus (BPIV-3) titre is 6.23 × 105.6TCID50Viral (BRSV) titre of/mL, Bovine Respiratory Syncytial for 8.9 × 106.8TCID50/ mL, bovine enteroviruses (BEV) titre are 4.1 × 107.8TCID50/ mL, bovine coronavirus (BcoV) titre are 5.8×106.5TCID50/ mL, bovine rota (BRV) titre are 2.3 × 107.0TCID50/ mL and bovine epizootic fever virus (BEFV) Titre is 3.2 × 105.5TCID50The cell culture poison of/mL, with reference to viral RNA extracts kit specification, extracts RNA, dissolving In 50 μ L RNAase-free water.According to Fermentas RevertAidTM First Strand cDNA Synthesis Kit specifications carry out RT-PCR reactions, obtain above-mentioned viral cDNA templates.
With 8.0 × 103TCID50The DNA of IBRV samples utilizes drawing for 1 optimal screening of embodiment as positive control template Thing and probe carry out RPA isothermal duplications to the cDNA of BVDV, BPIV-3, BRSV, BEV, BcoV, BRV and BEFV, and amplified production exists Sidestream chromatography test strips are detected.IBRV amplified productions show detection band and control in Sidestream chromatography test strips reaction zone Band, and shown clearly with other viral diagnosis sample test strips reaction zones only control line, detection line has no band, is feminine gender (Fig. 3).Illustrating the IBRV RPA amplification detection methods of the present invention has preferable specificity.
The repeatability investigation of 2.3 IBRV RPA amplification detection methods
With the 0.8TCID of extraction50The DNA of IBRV samples carries out RPA as template according to primer and probe described above Isothermal duplication, carries out 3 repetitions, amplified production is detected in Sidestream chromatography test strips respectively to the template.As a result phase is confirmed The amplified production of same amount template Sidestream chromatography test strips reaction zone show the identical detection band of brightness and control band, have compared with Good repeatability (Fig. 4).
Embodiment 3：Clinical sample IBRV is detected
The coarse extraction of 1.IBRV genomic DNAs
The nose that the established BVDV fluorescence quantitative RT-RCRs in this laboratory are accredited as the positive and negative oxen of IBRV is taken to wipe respectively Son amounts to 20 parts, adds 50 μ LTES (10mmolTris-HCl, pH8.0,5mmol/LEDTA, 0.5%SDS) and 150 μ g/mL eggs White enzyme K, acts on 15min, supernatant is taken, for RPA template amplifications by 65 DEG C.
2. clinical sample Sidestream chromatography RPA is detected
Using the genomic DNA of the clinical sample of preparation as template, according to the RPA described in step 1 in embodiment 2 Detection method is expanded.Expanded through RPA, hybrid product is shown in Sidestream chromatography test strips, 14 parts of sample test strips reaction zones Show control stripes band and test strip, show to contain IBRV in 14 parts of clinical samples, as a result method detections other with this experiment Coincidence rate is 100%, and the results are shown in Figure 5.
Embodiment 4：Kit for IBRV detections
It is prepared by 1.IBRV positive control templates
Upstream and downstream design primer (SEQ ID No.4 and the SEQ ID of the primer pair obtained respectively in the screening of embodiment 1 No.5), the DNA of the IBRV obtained using 2 step 1 of embodiment carries out PCR amplification as template, and reaction system is 50 μ L：10×LA 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 of template Each 2 μ L of upstream and downstream primer of L, 10 μm of ol/L, sterilizing ultra-pure water add to 50 μ L.Response procedures are 94 DEG C of pre-degeneration 3min, then Into 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, 31 circulations are carried out altogether；72 DEG C of extension 10min, finally stop at 4 DEG C.PCR is produced Thing screens recombinant plasmid, send through 1% agarose gel electrophoresis, recycling purpose segment rear clone to pEAST-T3 carriers, blue hickie Hua Da gene is sequenced, and sequencing result is compared, and correct recombinant plasmid is IBRV positive control templates.
2. the composition of kit：The primer and probe combination that embodiment 1 is screened, IBRV positive control templates, RPA amplifications Re Hydration buffer solutions, freeze enzyme granulate, magnesium acetate (280mM), deionized water and Sidestream chromatography test strips.
3. amplification system：Amplification system is 50 μ L, into the 0.2mL TwistAmp Exo reaction tubes containing lyophilized enzyme powder Add forward primer, each 2.1 μ L of reverse primer (10 μm of ol/L), 0.6 μ L of probe (10 μm of ol/L), 2 μ of IBRV positive control templates L, 29.5 μ LRehydration buffer solutions, 11.2 μ L deionized waters and 2.5 μ L magnesium acetate solutions (280mM).
4. detecting step：
The extraction of 4.1 viral DNAs
According to the IBRV DNA of the method extraction Clinical Processing sample of step 1 in embodiment 3.
4.2 RPA are expanded
It is anti-altogether in 38 DEG C of thermostat water baths according to the method progress RPA amplifications of " 1.2 IBRV RPA amplifications " in embodiment 2 Answer 25min.
4.3 Sidestream chromatography test strips are detected
Take 1 μ L amplified productions, the method for " 1.3 Sidestream chromatography test strips carry out amplified production detection " in Application Example 2 It is detected, if showing detection band and control band in Sidestream chromatography test strips, testing result is the positive, if test strips are only aobvious Show control band, then result is feminine gender.

Claims (9)

1. a kind of be used to combine by the primer and probe of RPA technology for detection infectious bovine rhinotrachetis viruses, it is characterised in that Its forward primer sequence is as shown in SEQ ID No.1, and reverse primer sequences are as shown in SEQ ID No.2, probe sequence such as SEQ Shown in ID No.3.
2. the primer and probe combination described in claim 1 is in the detection reagent for preparing detection infectious bovine rhinotrachetis virus Application.
3. a kind of kit for detecting infectious bovine rhinotrachetis virus, it is characterised in that the kit includes claim 1 institute The primer and probe combination stated.
4. kit as claimed in claim 3, it is characterised in that further included in kit：IBRV positive control templates, RPA amplifing reagents, deionized water and Sidestream chromatography detection reagent.
5. kit as claimed in claim 4, it is characterised in that the RPA amplifing reagents include：Rehydration is buffered Liquid, the recA recombinases of Escherichia coli, strand displacement archaeal dna polymerase, single-stranded DNA binding protein and magnesium acetate solution.
6. kit as claimed in claim 4, it is characterised in that Sidestream chromatography detection reagent include Sidestream chromatography test strips and Hybridization check buffer solution.
7. kit as claimed in claim 5, it is characterised in that the concentration of the magnesium acetate solution is 280mM.
8. kit as claimed in claim 4, it is characterised in that the preparation method of the IBRV positive control templates is：Point Not using primer of the sequence as shown in SEQ ID No.4 and SEQ ID No.5 as upstream and downstream primer, using the DNA of IBRV as template into Row PCR amplification, PCR product is through 1% agarose gel electrophoresis, recycling purpose segment rear clone to pEAST-T3 carriers, blue hickie Recombinant plasmid is screened, sequencing, sequencing result is compared, correct recombinant plasmid is IBRV positive control templates.
9. kit as claimed in claim 8, it is characterised in that the reaction system for carrying out the amplification of PCR is 50 μ L：10× LA PCR Buffer II5 μ L, 2.5 mM dNTP Mixture, 8 μ L, 0.5 μ L of LA Taq, template 2 μ L, 10 μm of ol/L's Each 2 μ L of upstream and downstream primer, sterilizing ultra-pure water add to 50 μ L；
The response procedures of PCR amplification are 94 DEG C of 3 min of pre-degeneration, common subsequently into 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s Carry out 31 circulations；72 DEG C of 10 min of extension, finally stop at 4 DEG C.
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CN106754934B (en) * 2016-11-28 2019-07-26 北京市农林科学院 A kind of aptamer of infectious bovine rhinotrachetis virus and application thereof
CN107557454A (en) * 2017-09-12 2018-01-09 苏州博尔达生物科技有限公司 Primer, probe, kit and its application based on RPA technology for detection chicken derived components
CN107557453A (en) * 2017-09-12 2018-01-09 苏州博尔达生物科技有限公司 Primer, probe, kit and its application based on RPA technology for detection pig derived components
CN107881259A (en) * 2017-11-15 2018-04-06 广西壮族自治区兽医研究所 A kind of pcr amplification primer thing of quick detection infectious bovine rhinotrachetis virus and its application
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CN108841926B (en) * 2018-07-13 2021-10-01 锦州医科大学 Primer, probe and kit for dual detection of hepatitis E virus and hepatitis A virus by RT-RPA-lateral flow chromatography
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