CN104862419A - Primer, probe and kit for detecting infectious bovine rhinotracheitis viruses - Google Patents
Primer, probe and kit for detecting infectious bovine rhinotracheitis viruses Download PDFInfo
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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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- 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 relates to a primer, a probe and a kit for detecting infectious bovine rhinotracheitis viruses, and discloses a primer and probe combination used for detecting the infectious bovine rhinotracheitis viruses according to an RPA technology, the forward primer sequence of the primer and probe combination is shown as SEQ ID No.1; the reverse primer sequence is shown as SEQ ID No.2; the probe sequence is shown as SEQ ID No.3. The invention further discloses the kit for detecting the infectious bovine rhinotracheitis viruses. The primer and the probe are adopted for detection, a clinical sample is only subjected to virus DNA crude extraction and RPA isothermal amplification, the result can be displayed on a lateral flow chromatography test strip, a heat circular reaction is not needed, and amplification needs not to be performed in a PCR instrument; the primer, the probe and the kit have the advantages of being high in sensitivity, strong in specificity, simple in reaction procedure, short in detection time, and suitable for clinical field detection in a non-lab environment, and have a wide application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of application recombinase polymeric enzymatic amplification technology (RecombinasePloymerase Amplification, RPA) and detect infectious bovine rhinotrachetis virus primer used and probe combinations and test kit thereof in conjunction with Sidestream chromatography technology (Lateral Flow Assay).
Background technology
Infectious bovine rhinotrachetis virus (Infectious bovine rhinotracheitis virus, IBRV) infectious bovine rhinotrachetis caused, that the one of ox is acute, hot, contagious disease, clinically in multiple types of presentation, particularly this disease can cause immunosuppression, and insecondary bacteriological infection can cause more serious respiratory tract disease.This disease is worldwide widely current at present, and this disease drastically influence international ox industry production trade, is classified as category-B transmissible disease by OIE (OIE).China detects IBRV in late 1970s from the milk cow of New Zealand's import, owing to there is no good immune protection measure, all there is this virus infection in the cows of China's most laboratories subsequently, on the fattening of cows, give milk and breed impact greatly, causing huge financial loss to cattle farm.At present pathogen separation, PCR method, ELISA test and serum neutralization test etc. are mainly contained to the diagnostic method of this disease, because these methods need accurate instrument and loaded down with trivial details testing sequence, be difficult to meet the requirement of Site Detection under non-lab environment.Thus need badly and set up a set of quick, accurate, easy diagnostic method, for clinical sites detection and epidemic monitoring etc. provide detection technique and the means of a new generation.
Recombinase polymeric enzymatic amplification (Recombinase Polymerase Amplification, RPA), be a kind of nucleic acid detection technique being different from PCR, mainly contain and participate in conjunction with the recombinase of single-chain nucleic acid, single-stranded DNA binding protein and strand displacement archaeal dna polymerase three kinds of enzymes.Its principle utilizes recombinase to be combined the Protein-DNA mixtures formed with primer, can find homologous sequence in double-stranded DNA.Under the help of single-stranded DNA binding protein, template DNA is unwind, and primer and template DNA start to match to be formed and copy 3 ' required C-terminal, carry out copying extension under the effect of archaeal dna polymerase, form new DNA complementary strand, exponential amplification is carried out to the target area in template.The design of primer sequence is most important to the result of RPA with selection.A biotin labeled reverse primer and the 5 ' fluorescently-labeled probe is added in RPA amplification system, there is an abasic site (dSpacer) at fluorophor 30 base places of probe mark, this site is the substrate of DNA repair enzyme effect, cutting can be identified by ribozyme nfo, produce 3 ' new C-terminal, carry out copying extension under the effect of archaeal dna polymerase, thus making the accumulated synchronized of two mark signal and amplified production, detected result can show in the Sidestream chromatography test strip that anti-FAM gold marks combination and antibiotin capture antibody.
Research at present for RPA technology is still in the starting stage, also RPA technology is not applied to the report that IBRV detects both at home and abroad.System of the present invention adopts RPA technology to set up the method for rapid detection IBRV first, and by specificity and sensitivity evaluation, can be used for clinical sites and detect, and the Site Detection for IBRV provides a kind of sensitive, reliable novel method.
Summary of the invention
Be applied to the blank of IBRV detection field for RPA technology, the invention provides the RPA detection method of a kind of accurate, quick, easy detection IBRV.Only need slightly extract by carrying out viral DNA to clinical sample and carry out RPA isothermal duplication, result can clear display in Sidestream chromatography test strip, do not need thermal cycle reaction, need not increase in PCR instrument, there is the advantages such as highly sensitive, high specificity, response procedures are simple, detection time is short, under being applicable to non-lab environment, clinical sites detects, and is with a wide range of applications.
For achieving the above object, the present invention adopts following technical proposals:
For passing through primer and a probe combinations of RPA technology for detection IBRV, its forward primer sequence is as shown in SEQ ID No.1, and reverse primer sequences is 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 '-hold mark with Biotin); (SEQ ID No.2)
Probe sequence: 5 '-CCGCAAAGTGCCGAGGGATGTCCTTGTAGT[dSpacer] (probe 5 '-hold marks with FAM CAGGTCCACCTTCCGCT-3 ', position about distance 5 '-end 30 bases substitutes a base with dSpacer, and 3 ' end C3-spacer blocks).
It should be noted that: react different from Standard PCR, the length of the required primer of RPA reaction is generally 30-35bp, the length of probe sequence is 46-52bp, during design of primers in order to avoid formed primer inner and between secondary structure, the increase of its length also makes design of primers and selects the increase of difficulty, therefore, primer design and select most important to the result of RPA.RPA technology is in starting conceptual phase, there is no special primer, probe design software, does not also have a large amount of data to provide foundation for its design of primers principle.Therefore, primer of the present invention and probe combinations need to design from target sequence two ends multipair primer to be optimized, to screen and just can obtain.
Present invention also offers a kind of test kit detecting IBRV, include for by the primer of RPA technology for detection IBRV and probe combinations in this test kit.
Further, also include in this test kit: IBRV positive control template, RPA amplifing reagent, deionized water and Sidestream chromatography detection reagent.IBRV positive control template, RPA amplifing reagent, deionized water form amplification system with primer together with probe combinations.
Described RPA amplifing reagent comprises Rehydration damping fluid, the recA recombinase of Escherichia coli, strand displacement archaeal dna polymerase, single-stranded DNA binding protein (SSB), 280mM magnesium acetate (MgAc) solution.
Described RPA Sidestream chromatography detection reagent comprises Sidestream chromatography test strip, hybridization check damping fluid (1 × PBS+0.1%Tween20).
The preparation method of described IBRV positive control template is the upstream and downstream design primer of the primer pair respectively shown in SEQ ID No.1 and SEQ ID No.2:
Upstream primer: 5 '-GCCAAGCGCAGCAGGCAGGTGAAT-3 ' (SEQ ID No.4);
Downstream primer: 5 '-CGACTGGGCGTACATCTCGGAGAA-3 ' (SEQ ID No.5);
With the DNA of IBRV for template carries out pcr amplification, reaction system is 50 μ L:10 × LA PCR Buffer II (Mg2+Plus) 5 μ L, 2.5mM dNTP Mixture 8 μ L, LA Taq 0.5 μ L (5U/ μ L), template 2 μ L, the each 2 μ L of upstream and downstream primer of 10 μm of ol/L, sterilizing ultrapure water adds to 50 μ L.Response procedures is 94 DEG C of denaturation 3min, then enters 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, carries out 31 circulations altogether; 72 DEG C extend 10min, finally stop at 4 DEG C.PCR primer is through 1% agarose gel electrophoresis, reclaim object segment rear clone to pEAST-T3 carrier, (be conventional carrier, commercialization buys) blue hickie screening recombinant plasmid, Hua Da gene is sent to check order, compared by sequencing result, correct recombinant plasmid is IBRV positive control template.
Concrete, a kind of test kit detecting IBRV, containing forward primer, each 2.1 μ L of reverse primer (10 μm of ol/L) in every 50 μ L amplification systems, probe 0.6 μ L (10 μm of ol/L), IBRV positive control template 2 μ L, Rehydration damping fluid 29.5 μ L, deionized water 11.2 μ L and magnesium acetate solution (280mM) 2.5 μ L.
The present invention also provides a kind of and applies RPA technology and in conjunction with the method for Sidestream chromatography technology for detection IBRV, step is as follows:
(1) the IBRV DNA that rapid extraction virus genom DNA test kit extracts cell culture and virus supernatant liquor is applied;
(2) according to IBRV conservative region design RPA Auele Specific Primer and probe, forward primer, each 2.1 μ L of reverse primer (10 μm of ol/L) are added in 50 μ L amplification systems of RPA amplification kit recommendation response, probe 0.6 μ L (10 μm of ol/L), template DNA 2 μ L, 29.5 μ LRehydration damping fluids, 11.2 μ L deionized waters and 2.5 μ L magnesium acetate solution (280mM), in the 0.2mL TwistAmp nfo reaction tubes containing lyophozyme powder, in thermostat water bath, 38 DEG C are reacted 25 minutes;
(3) get 1 μ L amplified production, application Sidestream chromatography test strip detects, if Sidestream chromatography test strip shows detection zone and contrast band, then detected result is positive, if test strip only shows contrast band, then result is negative.
Application aforesaid method can filter out a set of primer and the probe combinations that effectively can detect IBRV composition fast.
With known viruse titre for 8.0 × 10
7tCID
50after the IBRV deionized water of/mL carries out 10 times of serial dilutions, carry out synchronous detection after extracting DNA, result display can detect 0.8TCID
50target molecules, prove that the method has higher sensitivity.
With 8.0 × 10
3tCID
50the DNA of IBRV sample is as template, the primer of above-mentioned optimization and probe is utilized to carry out RPA isothermal duplication, amplified production shows detection zone and contrast band in Sidestream chromatography test strip, and all only show contrast band with the cDNA of other virus for template amplification, prove that the method has good specificity.
With the 0.8TCID extracted
50the DNA of IBRV sample, as template, carries out RPA isothermal duplication according to primer described above and probe, and carry out 3 times to this template and repeat, amplified production detects in Sidestream chromatography test strip respectively.Result confirms that the amplified production of identical amount template shows the identical detection zone of brightness and contrast band in Sidestream chromatography test strip reaction zone, has good repeatability.
Beneficial effect of the present invention:
(1) adopt primer of the present invention and probe combinations, method IBRV detected by RPA technology, there is higher sensitivity, specificity and repeatability.
(2) RPA technology of the present invention in conjunction with the method for Sidestream chromatography technology for detection IBRV, both can be used for the detection of the routine clinical samples such as clinical blood, milk sample, tissue, and also can detect trace samples such as Virus Aerosols.The present invention only need carry out viral genome to clinical sample and slightly extract, and constant-temperature amplification process, the amplification stage does not need special instrument, result is by vision visual inspection, do not need detector, and speed of response is fast, susceptibility is high, and the clinical sites that can complete IBRV in a non-laboratory environment in the short period of time detects.
Accompanying drawing explanation
The foundation of Fig. 1 IBRV diagnostic method, wherein, 1:RPA test kit provides primer, probe and template positive control; 2:IBRV negative control, template is H
2o; 3:IBRV genomic dna.
Fig. 2 sensitivity technique, being followed successively by virus titer from 1 to 10 templates is 8.0 × 10
7-8.0 × 10
-2tCID
50the DNA of IBRV sample.
The specific detection of Fig. 3 IBRV, wherein, 1: infectious bovine rhinotrachetis virus (IBRV); 2: Bovine Respiratory Syncytial 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 of Fig. 4 IBRV detects, and template is 0.8TCID
50the DNA of IBRV sample.
Fig. 5 clinical sample detects, wherein, and 1: positive control; 2: negative control; 3-16:IBRV positive sample; 17-22:IBRV negative sample.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, should be noted that following explanation is only to explain the present invention, not limiting its content.
Below in embodiment materials more used and reagent as follows: molecular biology reagents, TwistAmp nfo Kits is purchased from TwistDX company; Genline Hybridetect-1lateral flow strips is purchased from Milenia GmbH (Germany); LA TaqDNA Polymerase, viral DNA/RNA extract test kit purchased from precious biotechnology (Dalian) company limited; RevertAid
tMfirst Strand cDNA Synthesis Kit (catalog number (Cat.No.): K1622) is purchased from Fermentas company; PEASY-T3 CloningKit is purchased from Beijing Quanshijin Biotechnology Co., Ltd; Other biochemical reagents are import packing or domestic analytical pure.Primer and probe are synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
Instrument comprises: thermostat water bath, whizzer, vortex instrument, spectrophotometer and pure water instrument etc.
The experimental technique of unreceipted actual conditions in embodiment, usual condition conveniently, " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press of such as Sambrook etc., 2001) condition described in, or operate according to the condition that instrument or reagent manufacturer advise.
Embodiment 1: the design of primer and probe and screening
The design effectively of primer and probe is the most critical link determining Success in Experiment.But RPA technology is in starting conceptual phase, there is no special primer, probe design software, also do not have a large amount of data to provide foundation for its design of primers principle.Need to design multipair primer from target sequence two ends in current experiment to be optimized, screening, this technology design of primers requires extremely strict, and replacement or the increase and decrease of Individual base all can produce material impact to experimental result.Usually need during design to consider following factor: (1) primer length requires as 30-35bp, probe length requires, for 46-52bp, GC content is at 40%-60%, avoids primer inside to occur secondary structure and avoid primer to duplicate sequence.(2) detect amplified fragments and be less than 500bp.(3) avoid forming dimer between primer and probe, affect the accuracy of result.
Devise 4 probes according to the conserved sequence of the IBRV (accession number AJ004801.1) delivered in GenBank in this experiment, devise 3 upstream primers and 3 downstream primers in the both sides of every bar probe respectively.The genomic dna of cell culture and virus is extracted for template with viral DNA rapid extraction test kit, to the different primers of design, probe combinations carries out RPA amplification screening, be the primer of template to design with deionized water simultaneously, probe amplification makes negative control, with the template that RPA amplification kit provides, primer and probe combinations do positive control, finally select pair for amplification product and can show test strip and contrast band (see Fig. 1) clearly in Sidestream chromatography test strip, the primer filtered out and probe sequence are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 (see table 1).
Table 1 screens the primer and probe sequence that obtain
Note: probe 5 '-hold with FAM mark, the position about distance 5 ' end 30 bases substitutes a base with dSpacer, and 3 ' end C3-spacer blocks.
Biotin: vitamin H; FAM: luminophore; DSpacer: abasic site; C3-spacer: polymerase extension blocking group.
Embodiment 2: the foundation of laboratory IBRV RPA amplification detection method and investigation
1. the foundation of laboratory IBRV RPA amplification detection method
The extraction of 1.1 IBRV genomic dnas
Get 200 μ L cell culture and virus supernatant liquors, application rapid extraction virus genom DNA test kit extracts IBRV DNA, is finally dissolved in 50 μ L deionized waters.
1.2 RPA amplifications
Apply PRA method amplification IBRV distinguished sequence in the present embodiment, concrete steps are:
(1) in centrifuge tube, add each 2.1 μ L of upstream and downstream primer (10 μm of ol/L) that embodiment 1 designs, LF probe 0.6 μ L (10 μm of ol/L), Rehydration damping fluid 29.5 μ L, template DNA 2 μ L, uses ddH
2o supplies volume 47.5 μ L (table 2), of short duration centrifugal after whirlpool mixing;
Table 2RPA amplification system
(2) the above-mentioned mixed solution of 47.5 μ L is transferred in the 0.2mL TwistAmp nfo reaction tubes containing lyophozyme powder, repeatedly blow and beat with pipettor until whole grain dissolution;
(3) in each reaction tubes, add the magnesium acetate solution of 2.5 μ L (280mmol/L), the whirlpool that firmly turns upside down mixing 8-10 time, reaction occurs immediately;
(4) reaction tubes is put into the thermostat water bath of 38 DEG C, process 4min;
(5) react after 4min, take out reaction tubes, the whirlpool that firmly turns upside down mixing 8-10 time, of short duration centrifugal after put into 38 DEG C thermostat water bath continue to react 21min, obtain RPA reaction product.
1.3 Sidestream chromatography test strip carry out amplified production detection
Get the Sidestream chromatography test strip (dipstick, Milennia GenLine HybriDetect MGHD1) of some amount, detect catalogue number(Cat.No.) for difference and mark.Each detects sample and gets 99 μ L hybridization check damping fluids (HybriDetect AssayBuffer), adds in reaction tubes.Get 1 μ L hybridization product (RPA reaction product) to mix in centrifuge tube, hybridization reaction solution and hybridization check damping fluid be totally 100 μ L.The example reaction district of test strip is added reaction solution, hatches 5 minutes, after hatching end, from reaction solution, take out test strip, observe immediately.If test strip reaction zone shows two bands visible, then it is positive for detecting sample; If control line display in test strip reaction zone is clear, detection line has no band, then it is negative for detecting sample.
The investigation of 2.IBRV RPA amplification detection method
The susceptibility of 2.1 IBRV RPA amplification detection methods is investigated
Be 8.0 × 10 by known viruse titre
7tCID
50after the IBRV virus liquid deionized water of/mL carries out 10 times of serial dilutions, application virus genom DNA rapid extraction test kit extracts the DNA of virus, drawing 2 μ L IBRV DNA is respectively template, increase by " 1.2 primer amplification " working method, get 1 μ L hybridization product (RPA reaction product) to show in Sidestream chromatography test strip, result shows, can 0.8TCID be detected
50target molecules (Fig. 2), the primer of explanation the present invention design detects IBRV and has higher sensitivity and accuracy, and easy and simple to handle.
The specificity of 2.2 IBRV RPA amplification detection methods is investigated
Getting 200 μ L bovine viral diarrhea virus (BVDV) titres is 2.0 × 10
6.0tCID
50/ mL, bovine parainfluenza type-3 virus (BPIV-3) titre are 6.23 × 10
5.6tCID
50/ mL, Bovine Respiratory Syncytial virus (BRSV) titre are 8.9 × 10
6.8tCID
50/ mL, bovine enteroviruses (BEV) titre are 4.1 × 10
7.8tCID
50/ mL, bovine coronavirus (BcoV) titre are 5.8 × 10
6.5tCID
50/ mL, bovine rota (BRV) titre are 2.3 × 10
7.0tCID
50/ mL and bovine epizootic fever virus (BEFV) titre are 3.2 × 10
5.5tCID
50the cell cultures poison of/mL, extracts test kit specification sheets with reference to viral RNA, extracts RNA, be dissolved in 50 μ L RNAase-free water.According to Fermentas RevertAid
tMfirst Strand cDNASynthesis Kit specification sheets carries out RT-PCR reaction, obtains the cDNA template of above-mentioned virus.
With 8.0 × 10
3tCID
50the DNA of IBRV sample is as positive control template, and utilize the primer of embodiment 1 optimal screening and probe to carry out RPA isothermal duplication to the cDNA of BVDV, BPIV-3, BRSV, BEV, BcoV, BRV and BEFV, amplified production detects in Sidestream chromatography test strip.IBRV amplified production shows detection zone and contrast band in Sidestream chromatography test strip reaction zone, and only control line display is clear with other Viral diagnosis sample test strip reaction zone, and detection line has no band, is feminine gender (Fig. 3).Illustrate that IBRV RPA amplification detection method of the present invention has good specificity.
The repeatability of 2.3 IBRV RPA amplification detection methods is investigated
With the 0.8TCID extracted
50the DNA of IBRV sample, as template, carries out RPA isothermal duplication according to primer described above and probe, and carry out 3 times to this template and repeat, amplified production detects in Sidestream chromatography test strip respectively.Result confirms that the amplified production of identical amount template shows the identical detection zone of brightness and contrast band in Sidestream chromatography test strip reaction zone, has good repeatability (Fig. 4).
Embodiment 3: clinical sample IBRV detects
The thick extraction of 1.IBRV genomic dna
Get the BVDV fluorescence quantitative RT-RCR set up in this laboratory to be respectively accredited as IBRV nose swab that is positive and negative ox and to amount to 20 parts, add 50 μ LTES (10mmolTris-HCl, pH8.0,5mmol/LEDTA, 0.5%SDS) He 150 μ g/mL Proteinase Ks, 65 DEG C, effect 15min, get supernatant, for RPA template amplification.
2. clinical sample Sidestream chromatography RPA detects
Using the genomic dna of the clinical sample of preparation as template, increase according to the RPA detection method in embodiment 2 described in step 1.Increase through RPA, hybrid product is in the display of Sidestream chromatography test strip, and 14 this test strip of increment reaction zone display contrast band and test strips, show in 14 parts of clinical samples containing IBRV, result and this to test the coincidence rate that other method detects be 100%, result is as shown in Figure 5.
Embodiment 4: the test kit detected for IBRV
Prepared by 1.IBRV positive control template
Upstream and downstream design primer (SEQ ID No.4 and SEQ IDNo.5) of the primer pair obtained is screened respectively in embodiment 1, the DNA of the IBRV obtained with embodiment 2 step 1 carries out pcr amplification for template, reaction system is 50 μ L:10 × LA PCR Buffer II (Mg2+Plus) 5 μ L, 2.5mM dNTP Mixture 8 μ L, LA Taq 0.5 μ L (5U/ μ L), template 2 μ L, the each 2 μ L of upstream and downstream primer of 10 μm of ol/L, sterilizing ultrapure water adds to 50 μ L.Response procedures is 94 DEG C of denaturation 3min, then enters 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, carries out 31 circulations altogether; 72 DEG C extend 10min, finally stop at 4 DEG C.PCR primer, through 1% agarose gel electrophoresis, reclaims object segment rear clone to pEAST-T3 carrier, and blue hickie screening recombinant plasmid, send Hua Da gene to check order, compared by sequencing result, correct recombinant plasmid is IBRV positive control template.
2. the composition of test kit: the primer that embodiment 1 is screened and probe combinations, IBRV positive control template, RPA amplification Rehydration damping fluid, lyophozyme particle, magnesium acetate (280mM), deionized water and Sidestream chromatography test strip.
3. amplification system: amplification system is 50 μ L, forward primer, each 2.1 μ L of reverse primer (10 μm of ol/L) are added in the 0.2mL TwistAmp Exo reaction tubes containing lyophozyme powder, probe 0.6 μ L (10 μm of ol/L), IBRV positive control template 2 μ L, 29.5 μ LRehydration damping fluids, 11.2 μ L deionized waters and 2.5 μ L magnesium acetate solution (280mM).
4. detecting step:
The extraction of 4.1 viral DNAs
The IBRV DNA of Clinical Processing sample is extracted according to the method for step 1 in embodiment 3.
4.2 RPA amplifications
RPA amplification is carried out, coreaction 25min in 38 DEG C of thermostat water baths according to the method for " 1.2 IBRV RPA increase " in embodiment 2.
4.3 Sidestream chromatography test strip detect
Get 1 μ L amplified production, in Application Example 2, the method for " 1.3 Sidestream chromatography test strip carry out amplified production detection " detects, if Sidestream chromatography test strip shows detection zone and contrast band, then detected result is positive, if test strip only shows contrast band, then result is negative.
Claims (10)
1. one kind for by the primer of RPA technology for detection infectious bovine rhinotrachetis virus and probe combinations, it is characterized in that, its forward primer sequence is as shown in SEQ ID No.1, and reverse primer sequences is as shown in SEQ ID No.2, and probe sequence is as shown in SEQ IDNo.3.
2. primer according to claim 1 and probe combinations detect the application in the detection reagent of infectious bovine rhinotrachetis virus in preparation.
3. detect a test kit for infectious bovine rhinotrachetis virus, it is characterized in that, this test kit comprises primer according to claim 1 and probe combinations.
4. test kit as claimed in claim 3, is characterized in that, also include in test kit: IBRV positive control template, RPA amplifing reagent, deionized water and Sidestream chromatography detection reagent.
5. test kit as claimed in claim 4, it is characterized in that, described RPA amplifing reagent comprises: the recA recombinase of Rehydration damping fluid, Escherichia coli, strand displacement archaeal dna polymerase, single-stranded DNA binding protein and magnesium acetate solution.
6. test kit as claimed in claim 4, it is characterized in that, described RPA Sidestream chromatography detection reagent comprises Sidestream chromatography test strip and hybridization check damping fluid.
7. test kit as claimed in claim 5, it is characterized in that, the concentration of described magnesium acetate solution is 280mM.
8. test kit as claimed in claim 7, it is characterized in that, amplification system is 50 μ L, forward primer, each 2.1 μ L of reverse primer are added in the 0.2mL TwistAmp nfo reaction tubes containing lyophozyme powder, probe 0.6 μ L, IBRV positive control template 2 μ L, deionized water 11.2 μ L and magnesium acetate solution 2.5 μ L.
9. test kit as claimed in claim 4, it is characterized in that, the preparation method of described IBRV positive control template for: respectively with the primer of sequence as shown in SEQ ID No.4 and SEQ ID No.5 for upstream and downstream primer, with the DNA of IBRV for template carries out pcr amplification, PCR primer, through 1% agarose gel electrophoresis, reclaims object segment rear clone to pEAST-T3 carrier, blue hickie screening recombinant plasmid, order-checking, compared by sequencing result, correct recombinant plasmid is IBRV positive control template.
10. test kit as claimed in claim 9, it is characterized in that, the reaction system of carrying out the amplification of PCR is 50 μ L:10 × LAPCR Buffer II5 μ L, 2.5 mM dNTP Mixture 8 μ L, LA Taq 0.5 μ L, template 2 μ L, the each 2 μ L of upstream and downstream primer of 10 μm of ol/L, sterilizing ultrapure water adds to 50 μ L;
The response procedures of pcr amplification is 94 DEG C of denaturation 3min, then enters 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, carries out 31 circulations altogether; 72 DEG C extend 10min, finally stop at 4 DEG C.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132590A (en) * | 2015-10-15 | 2015-12-09 | 上海市农业科学院 | LAMP visual detection method of infectious bovine rhinotracheitis viruses |
| CN106754934A (en) * | 2016-11-28 | 2017-05-31 | 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 |
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| CN107988434A (en) * | 2017-12-22 | 2018-05-04 | 西南民族大学 | Infectious bovine rhinotrachetis virus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
| CN108841926A (en) * | 2018-07-13 | 2018-11-20 | 锦州医科大学 | A kind of primer, probe and the kit of RT-RPA- Sidestream chromatography double check Hepatitis E virus and hepatitis A virus |
| CN111560471A (en) * | 2020-04-26 | 2020-08-21 | 北京市动物疫病预防控制中心 | Nucleic acid, kit and micro-drop digital PCR method for detecting infectious bovine rhinotracheitis virus |
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| CN116377133A (en) * | 2023-03-01 | 2023-07-04 | 宁夏大学 | Primer, kit and application for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus |
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| CN105132590B (en) * | 2015-10-15 | 2022-02-08 | 上海市农业科学院 | LAMP visual detection method of infectious bovine rhinotracheitis virus |
| CN105132590A (en) * | 2015-10-15 | 2015-12-09 | 上海市农业科学院 | LAMP visual detection method of infectious bovine rhinotracheitis viruses |
| CN106754934A (en) * | 2016-11-28 | 2017-05-31 | 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 |
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| CN107988434A (en) * | 2017-12-22 | 2018-05-04 | 西南民族大学 | Infectious bovine rhinotrachetis virus detection kit and application based on constant temperature isolation type fluorescent PCR platform |
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| CN111560471A (en) * | 2020-04-26 | 2020-08-21 | 北京市动物疫病预防控制中心 | Nucleic acid, kit and micro-drop digital PCR method for detecting infectious bovine rhinotracheitis virus |
| CN113278736A (en) * | 2021-05-28 | 2021-08-20 | 深圳市易瑞生物技术股份有限公司 | Reagent and method for qualitative detection of bovine herpes virus type I |
| CN116254373A (en) * | 2023-03-01 | 2023-06-13 | 宁夏大学 | RPA primer pair, kit and detection method for detecting bovine rhinotracheitis virus |
| CN116377133A (en) * | 2023-03-01 | 2023-07-04 | 宁夏大学 | Primer, kit and application for dual RPA amplification of infectious bovine rhinotracheitis virus and bovine viral diarrhea virus |
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