CN106834549A - The cross primer amplification immune chromatography test paper of detection pseudorabies virus street strain is combined the primer and probe groups and kit of method - Google Patents
The cross primer amplification immune chromatography test paper of detection pseudorabies virus street strain is combined the primer and probe groups and kit of method Download PDFInfo
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Abstract
The invention discloses primer and probe groups and kit that a kind of cross primer amplification immune chromatography test paper for detecting pseudorabies virus street strain is combined method.The cross primer that the present invention is disclosed for detecting pseudorabies virus street strain first expands the primer and probe groups of immune chromatography test paper combination method, including two Outside primers, a cross primer and two probes.The cross primer amplification immune chromatography test paper combination kit of further open detection pseudorabies virus street strain of the invention, including cross primer amplifing reagent and immuno-chromatographic test paper strip.The present invention sets up CPA reaction systems and is expanded with virus genom DNA as template using cross primer amplifing reagent, and amplified production loading immuno-chromatographic test paper strip carries out result judgement.Primer of the present invention and probe groups and kit energy specific detection pseudorabies virus street strain, Sensitivity and Specificity are good, and visualization is high, easy to operate, is suitable to now detecting for pseudorabies virus.
Description
Technical field
The present invention relates to the detection of pseudorabies virus street strain, more particularly to the friendship for detecting pseudorabies virus street strain
Fork primer amplification-test strips are combined the primer and probe groups and kit of method, belong to the detection of pseudorabies virus street strain
Field.
Background technology
Pseudoabies (Pseudorabies, PR) is caused by pseudorabies virus (Pseudorabies virus, PRV)
Can infected pigs and other many animals acute infectious disease.The disease can cause piglet death, shelf porcine respiratory disease, sow
(Mettenleiter, T.C., 1996.Immunobiology of the pseudorabies (Aujeszky ' s such as miscarriage
disease).Vet.Immunol.Immunopathol.54,221–229.).PRV is also referred to as Ao Yecijishi viruses or porcine herpe
Viral 1 type, category herpetoviridae, full-length genome 143kb (Nauwynck, H., Glorieux, S., Favoreel.H.,
Pensaert,M.,2007.Cell biological and molecular characteristics of
pseudorabies virus infections in cell cultures and in pigs with emphasis on
the respiratory tract.Vet.Res.38,229–241；Mettenleiter,T.C.,2000.Aujeszky’s
disease(Pseudorabies)virus:the virus and molecular pathogenesis–state of the
art.Vet.Res.31,99–115.).Bartha-K61 is a kind of PRV vaccines of gE/gI gene delections, can be resisted for pig
PRV provides protection.
Since the end of the year 2011, pseudoabies is broken out on the pig farm in China's multiple province successively, even if Bartha- was immunized
K61 attenuated vaccines, still cannot effectively resist new epidemic situation.Research confirms that this time epidemic situation is caused by PRV variants, meanwhile, mesh
Preceding Bartha-K61 vaccine strains be not highly resistant to PRV variants (An, T.Q., Peng, J.M., Tian, Z.J., Zhao,
H.Y.,Li,N.,Liu,Y.M.,Chen,J.Z.,Leng,C.L.,Sun,Y.,Chang,D.,Tong,G.Z.,
2013.Pseudorabies virus variant in Bartha-K61-vaccinated pigs,China,
2012.Emerg.Infect.Dis.19,1749-1755.), thus the virus diagnosis detection turn into control the epidemic situation it is main
Means.
PRV classical detection method for immunofluorescent test (Stewart, W.C., Carbrey, E.A., Kresse,
J.I.,1967.Detection of pseudorabies virus by
immunofluorescence.J.Am.Vet.Med.Assoc.151,747–751；Ducatelle,R.,Coussement,W.,
Hoorens,J.,1982.Immunoperoxidase study of Aujeszky’s disease virus in
Pigs.Res.Vet.Sci.32,294-302.) and virus purification (Pensaert, M.B., Kluge, J.P.,
1989.Pseudorabies virus(Aujeszky’s disease).In:Pensaert MB(ed)Virus
Infections of Porcines.Elsevier Press, Amsterdam, pp 39-65.), but both approaches are tested
Excessive cycle.At present, various PRV have been set up often with detection method, such as polymerase chain amplified reaction (PCR), quantitative fluorescent PCR,
Immune chromatography test paper and the electrochemical reaction based on collaurum.But, serological analysis method is difficult to detection PRV infection early stages
Positive sample, and molecule diagnostic analysis method generally need complexity specific apparatus.Accordingly, it would be desirable to set up a kind of quick, steady
It is fixed, be used to monitor this epidemic situation without complex instrument and the diagnostic method that can carry out now detecting.
Cross primer amplification (CPA) technology be developed in recent years a kind of new diagnosis detecting method (Xu, G., Hu,
L.,Zhong,H.,Wang,H.,Yu,S.,Weiss,T.C.,Romaniuk,P.L.,You,Q.,2012.Cross Priming
Amplification:Mechanism and Optimization for Isothermal DNA
Amplification.Sci.Rep.2,246–252.).The amplification system of the method is handed over by four to five general primers and one
Fork primer composition, under constant temperature, generates the amplified production of hair fastener type different in size.CPA amplified productions can with it is special
With reference to DNA double chain fluorescent dye combine, send fluorescence, judged according to fluorescence intensity, but fluorescence signal be difficult to it is naked
Eye is accurately identified.
Therefore, set up a kind of cross primer amplification-immune chromatography test paper and be combined method (CPA-strip) for detecting PRV open countries
Poison, with reference to the efficient DNA cloning abilities of CPA and test strips visualization feature, will be with important meaning for effectively control PR epidemic situations
Justice.
The content of the invention
First technical problem to be solved by this invention is to provide the intersection for detecting pseudorabies virus street strain
The primer and probe groups of primer amplification-immune chromatography test paper combination method；
Second technical problem to be solved by this invention is to provide a kind of intersection for detecting pseudorabies virus street strain
Primer amplification-immune chromatography test paper is combined kit, and sets up a kind of cross primer expansion for detecting pseudorabies virus street strain
Increasing-immune chromatography test paper is combined method.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention discloses the cross primer amplification-immune chromatography test paper for detecting pseudorabies virus street strain first
The primer and probe groups of combination method, including two Outside primers, a cross primer and two probes；
Wherein, the nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.1, the nucleotides of the Outside primer 2
Sequence is shown in SEQ ID No.2；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.3；The core of the probe 1
Nucleotide sequence is that the nucleotides sequence of the probe 2 is classified as (i.e. scheme 3) shown in SEQ ID No.5 shown in SEQ ID No.4；Or
Person,
The nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.6, the nucleotide sequence of the Outside primer 2
Shown in SEQ ID No.7；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.8；The nucleotides of the probe 1
Sequence is that the nucleotides sequence of the probe 2 is classified as (i.e. scheme 1) shown in SEQ ID No.10 shown in SEQ ID No.9；Or,
The nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.11, the nucleotide sequence of the Outside primer 2
Shown in SEQ ID No.12；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.13；The nucleosides of the probe 1
Acid sequence is that the nucleotides sequence of the probe 2 is classified as (i.e. scheme 2) shown in SEQ ID No.15 shown in SEQ ID No.14.
Most pseudoabies (PR) vaccine is gE/gI deletion of vaccine, such as Bartha-K61, SA215 and HB98.This hair
It is bright to analyze PRV difference strain gene orders, the conserved region of the gI/gE genes for strategically being lacked in Bartha-K61 vaccine strains
Multigroup primer and probe are designed in domain, respectively including two Outside primers, two probes and a cross primer.Probe and primer
The selection result shows that the scheme 1 and scheme 2 designed by the present invention may be produced when negative sample is detected with pig genomic DNA
Raw cross reaction, produces false positive, it is impossible to accurately detect PRV；And scheme 3 is generated when negative sample is detected without band, with pig
Genomic DNA no cross reaction, while there is obvious band to generate when positive is detected.The present invention by the middle probe of scheme 3 and
Primer is compared with known PRV strains sequence in NCBI, as a result shows that probe and primer can correctly recognize known array.Cause
This, the primer and probe of selection scheme of the present invention 3, i.e., the nucleotides sequence of described Outside primer 1 are classified as shown in SEQ ID No.1,
The nucleotides sequence of the Outside primer 2 is classified as shown in SEQ ID No.2；The nucleotides sequence of the cross primer is classified as SEQ ID
Shown in No.3；The nucleotides sequence of the probe 1 is classified as shown in SEQ ID No.4, and the nucleotides sequence of the probe 2 is classified as SEQ ID
Shown in No.5.
Primer of the present invention and probe groups can be applied to detection pseudorabies virus street strain, enable in particular to application
Method is combined in the cross primer amplification-immune chromatography test paper for setting up detection pseudorabies virus street strain.
Pseudorabies virus street strain of the present invention, i.e. wild type PRV strains.
The present invention further discloses a kind of cross primer amplification-immunity-chromatography test for detecting pseudorabies virus street strain
Paper is combined method, comprises the following steps：(1) virus genom DNA is extracted from detected sample；(2) it is mould with the DNA for extracting
Plate, sets up CPA reaction systems and is expanded with primer of the present invention and probe groups；(3) amplified production loading is immunized
Chromatograph test strip, if developed the color at the detection line and nature controlling line of immuno-chromatographic test paper strip, result judgement (judges for the positive
Virus to be detected is pseudorabies virus street strain)；If developed the color only at nature controlling line, result judgement (judges for feminine gender
Viral Bu Shi pseudorabies viruses street strain to be detected).
It is further preferred that a kind of cross primer amplification-immune chromatography test paper connection for detecting pseudorabies virus street strain
Usage, comprises the following steps：(1) virus genom DNA is extracted from detected sample；(2) it is template with the DNA for extracting, with
Nucleotides sequence is classified as the Outside primer 1 shown in SEQ ID No.1, the Outside primer 2 shown in SEQ ID No.2, SEQ ID No.3
Probe 1 shown in shown cross primer, SEQ ID No.4 and the probe 2 shown in SEQ ID No.5 set up CPA reaction systems
And expanded；(3) by amplified production loading immuno-chromatographic test paper strip, if the detection line and nature controlling line of immuno-chromatographic test paper strip
Place develops the color, then result judgement is positive (judging that virus to be detected is pseudorabies virus street strain)；If only Quality Control
Developed the color at line, then result judgement is feminine gender.
Wherein, the CPA reaction systems include：The cumulative volume of reaction system is 25.0 μ L；Wherein, 10 μM of cross primers
2.0 μ L, 10 μM of the μ L of Outside primer 1 0.5,10 μM of the μ L of Outside primer 2 0.5,10 μM of the μ L of probe 1 1.0,10 μM of μ of probe 2 1.0
L、10mM dNTP 2.5μL、100mM Mg2+1.0 μ L, the μ L of 10 × ThermoPol buffer solutions 2.5, the Bst DNA polymerizations of 8U/ μ L
The μ L of enzyme 1.0, the μ L of genomic DNA 2.0, the amplification μ L of reinforcing agent 2.5, with deionized water polishing to 25.0 μ L.The amplification reinforcing agent
Selected from glycine betaine or dimethyl sulfoxide (DMSO), preferably glycine betaine.The program of the amplification is：59 DEG C of -67 DEG C of constant-temperature amplification 60min；
Preferably, 61 DEG C of constant-temperature amplification 60min.
It is described in the cross primer amplification-immune chromatography test paper combination method of present invention detection pseudorabies virus street strain
The 5 ' of probe 1-end is connected with FITC (fluorescein isothiocynate, fluorescein isothiocyanate), the probe 2
5 '-end is connected with biotin；Preferably, nucleotides sequence be classified as probe 1 shown in SEQ ID No.4 5 '-end be connected with FITC；Core
Nucleotide sequence is connected with biotin for 5 '-end of probe 2 shown in SEQ ID No.5.
It is described in the cross primer amplification-immune chromatography test paper combination method of present invention detection pseudorabies virus street strain
Immuno-chromatographic test paper strip includes：Sample pad, pad, detection line, nature controlling line, adsorptive pads and PVC board；Wherein, the detection line
On be coated with antibiotin monoclonal antibody, mountain sheep anti-mouse igg is coated with nature controlling line；It is anti-containing pan coating in pad
Gold nano grain (the method bibliography of gold nano grain pan coating anti-FITC monoclonal antibody of FITC monoclonal antibodies
Zhou,Y.,Pan,F.G.,Li,Y.S.,Zhang,Y.Y.,Zhang,J.H.,Lu,S.Y.,Ren,H.L.,Liu,Z.S.,
2009.Colloidal gold probe-based immunochromatographic assay for the rapid
detection of brevetoxins in fishery product samples.Biosens.Bioelectron.24,
2744–2747.)。
In order to improve the amplification efficiency and sensitiveness of CPA reactions, the present invention need to be carried out to reaction temperature and amplification reinforcing agent
Optimization.Result shows, under identical temperature conditionss, when reaction system adds glycine betaine, the amplified production of generation is most.Together
When, when reaction temperature is 61 DEG C, amplified production content is apparently higher than the amplified production content under the conditions of other reaction temperatures.Cause
This, present invention determine that CPA optimum reaction conditions are：Reaction temperature is 61 DEG C, while it is glycine betaine to expand reinforcing agent.
The invention also discloses a kind of cross primer amplification-immune chromatography test paper connection for detecting pseudorabies virus street strain
With kit, including：Cross primer amplifing reagent and immuno-chromatographic test paper strip；Wherein, the cross primer amplifing reagent includes：
Outside primer 1, Outside primer 2, cross primer, probe 1, probe 2, dNTP, Mg2+, 10 × ThermoPol buffer solutions, Bst DNA
Polymerase and amplification reinforcing agent；The Outside primer 1, Outside primer 2, cross primer, probe 1 and probe 2 are selected from institute of the present invention
The primer and probe groups stated, 5 '-end of the probe 1 are connected with FITC, and 5 '-end of the probe 2 is connected with biotin；It is preferred that
, the nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.1, and the nucleotides sequence of the Outside primer 2 is classified as SEQ
Shown in ID No.2；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.3；The nucleotides sequence of the probe 1 is classified as
Shown in SEQ ID No.4, the nucleotides sequence of the probe 2 is classified as shown in SEQ ID No.5；The amplification reinforcing agent is selected from beet
Alkali or dimethyl sulfoxide (DMSO), preferably glycine betaine.The immuno-chromatographic test paper strip includes：Sample pad, pad, detection line, Quality Control
Line, adsorptive pads and PVC board；Wherein, antibiotin monoclonal antibody is coated with the detection line, is coated with the nature controlling line
Mountain sheep anti-mouse igg, contains the gold nano grain of pan coating anti-FITC monoclonal antibody in the pad.
The CPA reaction systems set up using the cross primer amplifing reagent described in kit of the present invention are included：Reactant
The cumulative volume of system is 25.0 μ L；Wherein, the 10 μM of μ L of cross primer 2.0,10 μM of the μ L of Outside primer 1 0.5,10 μM of Outside primers 2
0.5 μ L, 10 μM of the μ L of probe 1 1.0,10 μM of the μ L of probe 2 1.0, the μ L of 10mM dNTP 2.5,100mM Mg2+1.0μL、10×
The μ L of ThermoPol buffer solutions 2.5, the μ L of Bst archaeal dna polymerases 1.0 of 8U/ μ L, the μ L of genomic DNA 2.0, amplification reinforcing agent 2.5 μ
L, with deionized water polishing to 25.0 μ L.The program expanded using the cross primer amplifing reagent is：59 DEG C of -67 DEG C of perseverances
Temperature amplification 60min；Preferably, 61 DEG C of constant-temperature amplification 60min.
Specific detection result shows that the cross primer of the detection pseudorabies virus street strain that the present invention is set up expands
Increasing-immune chromatography test paper is combined the method swine disease virus no cross reaction common with CSFV, PCV2, PPV and PRRSV etc., can
Specific detection PRV street strains (including BJKJZ2015, HeBLP2014, HLJMDJ2013, TJ plant of PRV variants, PRV classics
Strain Min-A, SC strain), it is impossible to detect PRV vaccine strains Bartha-K61, HB98 and SA-215.Sensitivity analysis result shows,
The lowest detection of the inventive method is limited to 200 copies.Repeated testing result shows, can know without naked eyes at same concentration detection line
Other color change.Accordance analysis result shows, the coincidence rate of CPA-strip methods of the present invention and triple fluorescent quantitative PCR
It is 100%, is 98.2% with the coincidence rate of virus purification；Wherein, 6 Bartha-K61 vaccine strain positives, through the present invention
CPA-strip methods are detected as feminine gender.Result above shows, the intersection of the detection pseudorabies virus street strain that the present invention sets up
Primer amplification-immune chromatography test paper is combined method, with good sensitiveness, specificity and effect of visualization, can be used in PRV's
Now detect.
At present, although have some detection methods for PRV, such as LAMP (Zhang, C.F., Cui, S.J., Zhu, C.,
2010.Loop-mediated isothermal amplification for rapid detection and
differentiation of wild-type pseudorabies and gene-deleted virus
Vaccines.J.Virol.Methods 169,239-243.) and immune chromatography test paper (Guo, D.L., Pan, Q.W., Li,
K.P.,Li,J.Q.,Shen,H.W.,Wang,X.L.,Zhang,X.Y.,Li,X.S.,Fu,F.,Feng,L.,Li,X.,
2015.Development and clinical evaluation of a new gold-immunochromatographic
assay for the detection of antibodies against field strains of pseudorabies
Virus.J.Virol.Methods 222,164-169.), but these diagnosis detecting methods respectively have shortcoming.Early stage is infected in PRV,
Pig body needs a period of time to produce the antibody of anti-PRV.The clinical practice result of immune chromatography test paper shows that the method earliest can only
The antibody for detecting anti-PRV on the 13rd day after Infected pig PRV.Therefore, immune chromatography test paper is difficult to infect early stage sample to PRV
Product are detected.Molecule diagnostic analysis method, such as LAMP and fluorescence quantifying PCR method, it usually needs complicated instrument, it is difficult to enter
Row now is detected.In addition, conventional isothermal amplification technology, its amplified production is combined with fluorescent dye, sends fluorescence, or with it is supporting
Reagent reacting generates magnesium pyrophosphate white precipitate；But its result visualization low degree, it is difficult to the naked eye judge, simultaneous reactions mistake
Primer dimer in journey is likely to influence the judgement of result.
Cross primer amplification-immune chromatography test paper combination (CPA-strip) method that the present invention sets up, respectively in two spies
The 5 ' of pin-end is connected with biotin and FITC, CPA are reacted after terminating, amplified production two ends difference flag F ITC and biotin, shape
Into biotin-dsDNA-FITC compounds, as a new antigen, recognized by test strips jointly, can priority and colloid golden watch
Anti-biotin antibodies at face anti-FITC antibody and detection line are combined, and form compound, and collaurum is fixed at detection line, are examined
Developed the color at survey line, result judgement is the positive, visualization is high.Meanwhile, the primer dimer in course of reaction is sentenced to result
Determine without influence.Based on molecule diagnostic analysis, the inventive method is applied to the sample that PRV infects early stage.Although CPA- of the present invention
The sensitiveness of strip methods is slightly below LAMP (100 copy) (Zhang et al., 2010) and triple fluorescent quantitative PCR method
(50 copy) (Meng, X.Y., Luo, Y., Liu, Y., Shao, L., Sun, Y., Li, Y., Li, S., Ji, S., Qiu, H.J.,
2016.A triplex real-time PCR for differential detection of classical,variant
And Bartha-K61vaccine strains of pseudorabies.Arch.Virol.161,2425-2430.), but
The inventive method is easy to operate, with good effect of visualization, can carry out now detecting.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention strategically draws in two outsides of conserved regions design of the gI/gE genes of Bartha-K61 vaccine strains missing
Thing, two probes and a cross primer, the cross primer of the detection pseudorabies virus street strain for being set up expands-layer is immunized
Analysis test paper combination method, can specifically detect PRV street strains, and cannot detect the vaccines such as Bartha-K61, SA215 and HB98
Strain.The inventive method combines the efficient DNA cloning abilities of CPA and test strips visualization feature, without special or complex instrument,
Reliability easy to operate, visualization is high, it is easy to judge, can carry out now detecting, it is adaptable to PR epidemic diseases increasingly serious at present
Feelings, for the diagnosis and prevention and control of PRV provide effective detection means, with application value very high.
Brief description of the drawings
Fig. 1 is the design attitude of primer and probe；
Fig. 2 is CPA-strip detection method principle schematics；Wherein, Gold nanoparticles are gold nano grain；
Biotin is biotin；Product of CPA are CPA products；Monoclonal antibodies against biotin are
The monoclonal antibody of antibiotin；Monoclonal antibodies against FITC are anti-FITC monoclonal antibody；
Goat anti-mouse IgG antibody are the IgG of mountain sheep anti mouse；Sample pad are sample pad；Conjugated pad
It is pad；Test line are detection line；Control line are nature controlling line；PVC sheet are PVC board；Absorbent
Pad is adsorptive pads；
Fig. 3 is the selection of CPA-strip primers and probe；Wherein, A-C is respectively scheme 1 to the amplification of scheme 3；
M：DNA marker；N：Negative control；P：Positive control；1-5：Negative sample；
Fig. 4 is the sequence alignment of primer and probe in NCBI in scheme 3；Wherein, it is PRV classics strains and variation in dashed box
The primer and probe design attitude of strain；Shi Kuang mesogen strains are PRV variants, and other strains are classical strain；Identical sequence () table
Show；
Fig. 5 synthesizes for mass spectral analysis detection probe；Wherein, A：PRV-3s probes；B：PRV-3s general primers；C：PRV-1s
Probe；D：PRV-1s general primers；
Fig. 6 is the selection of CPA reaction temperatures and amplification reinforcing agent；Wherein, M：DNA marker；1-5：59℃、61℃、63
℃、65℃、67℃；6：Negative control；7-11：59℃、61℃、63℃、65℃、67℃；12：Negative control；DMSO is diformazan
Base sulfoxide；
Fig. 7 is CPA-strip methods detection PRV difference strains；Wherein, Negative control are negative control；
Fig. 8 is the specificity analysis of CPA-strip methods；Wherein, Negative control are negative control；
Fig. 9 is the sensitiveness of CPA-strip methods；Wherein, test paper 1：Negative control；Test paper 2~10：Plasmid standard point
Wei 2~2.0 × 108Individual copy.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any limitation is not constituted to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications or replacement each fall within protection scope of the present invention.
Cross primer amplification-the immune chromatography test paper of the detection PRV of embodiment 1 street strains is combined building for method (CPA-strip)
It is vertical
1st, material and method
1.1 viruses and sample
Three class PRV strains be used for detect, respectively classical strainses Min-A and SC, variation strain HLJMDJ2013,
BJKJZ2015, HeBLP2014 and TJ, vaccine strain Bartha-K61, SA215 and HB98.Other viruses for being used to detect include pig
Pestivirus (Classical swine fever virus, CSFV) Strain Shimen, pig parvoviral (Porcine parvovirus,
PPV) Z plants, JXL plants of porcine circovirus 2 type (Porcine circovirus type 2, PCV2) and pig breeding are comprehensive with breathing
Levy virus (Porcine reproductive and porcine respiratory syndrome virus, PRRSV) HuN4
220 samples that 2013-2016 China difference province pig farm is collected in strain altogether carry out accordance experiment.
The selection of 1.2 primers and probe
NCBI websites Blast analysis PRV difference strain gene orders, in the gI/gE bases of Bartha-K61 vaccine strains missing
Because multigroup primer and probe (Fig. 1 and Biao 1) are designed in region.All primers and probe are all in Shanghai life work synthesis.To probe and drawing
Thing is selected, and experiment condition is as follows：The μ L of PRV-1s-2a 2.0 (10 μM), each 0.5 μ L of PRV-4a and PRV-5s (10 μM),
Each 1.0 μ L of PRV-3s and PRV-1s (10 μM), the μ L (10mM) of dNTP 2.5, Mg2+1.0 μ L (100mM), ThermoPol buffer solutions
The μ L of (10 ×) 2.5 μ L, Bst archaeal dna polymerase 1.0 (8U/ μ L), the μ L of genomic DNA 2.0, amplification the μ L of reinforcing agent 2.5, spend from
Sub- water polishing is to 25.0 μ L.The constant-temperature amplification 60min at 61 DEG C.Product detected with 1.5% agarose gel electrophoresis,
It is determined that optimal primer and probe.The primer and probe application that will be selected are in subsequent experimental.
The primer and probe implementation sequence of the CPA-strip of table 1
The extraction of 1.3 virus genom DNAs
Using DNA extraction kit (Omega, the U.S.), according to operating instruction extract virus infection cell culture or
Organize the genomic DNA of homogenate.
The preparation of 1.4 PRV plasmid templates DNA
The pcr amplification product that primer PRV-4a and PRV-5s are obtained is cloned into pMD-18T carriers, obtains pMD-18T-
PRV templates, as the standard items of detection.By recombinant plasmid from 2.0 × 108Copy/μ L are diluted to 2 copies/μ L, -20 DEG C of storages
It is standby.
The foundation and optimization of 1.5 PRV-CPA reaction systems
Probe and primer to selecting carry out condition optimizing, and reaction system is as follows：PRV-1s-2a 2.0μL(10μM)、
Each 0.5 μ L of PRV-4a and PRV-5s (10 μM), each 1.0 μ L of PRV-3s and PRV-1s (10 μM), the μ L (10mM) of dNTP 2.5, Mg2+
1.0 μ L (100mM), the μ L (10 ×) of ThermoPol buffer solutions 2.5, the μ L of Bst archaeal dna polymerases 1.0 (8U/ μ L), genomic DNA
2.0 μ L and the amplification μ L of reinforcing agent 2.5 [glycine betaine or dimethyl sulfoxide (DMSO) (DMSO)], with the μ L of deionized water polishing 25.0.At 59 DEG C
At to 67 DEG C 60min is expanded under constant temperature.Product is detected with 1.5% agarose gel electrophoresis.It is determined that optimal anti-
Condition is answered for following experiment.
The preparation of 1.6 gold nano grain conjugates and immuno-chromatographic test paper strip
According to reported document (Zhou, Y., Pan, F.G., Li, Y.S., Zhang, Y.Y., Zhang, J.H., Lu,
S.Y.,Ren,H.L.,Liu,Z.S.,2009.Colloidal gold probe-based immunochromatographic
assay for the rapid detection of brevetoxins in fishery product
Samples.Biosens.Bioelectron.24,2744-2747.), in gold nano grain (AuNP) pan coating anti-FITC list
Clonal antibody (McAb).Use 0.2M K2CO3The pH of AuNP solution is adjusted to 8.4.The anti-FITC McAb that about 10 μ g are purified is added
To in 1mL AuNP solution, and mix 20min at 25 DEG C.Then, it is slowly added to 1% (w/v) BSA and stirs 20min, 600
× g centrifugations 25min removes precipitation.By supernatant is in 11000 × g centrifugation 25min and retains precipitation.AuNP conjugates are with 800 μ L
Buffer solution [containing 1% (w/v) BSA, 0.1% (v/v) Tween-20,0.02% (w/v) sodium azide and 2.4% (w/v)
Tris-HCl (pH=8.4)] it is resuspended.Finally, AuNP conjugates are added in pad, 2h is dried at 7 DEG C.Antibiotin
The IgG (1mg/mL) of McAb (1mg/mL) and mountain sheep anti mouse is fixed on NC films respectively as detection line and nature controlling line with 2 μ L/cm
On.Stored under 4 DEG C of drying conditions.Test strips are standby by Hangzhou You Sida company systems.
1.7 CPA-strip detect various PRV strains
Detect various PRV strains using the CPA-strip for setting up, including wild type PRV strains Min-A, SC, TJ,
HLJMDJ2013, BJKJZ2015 and HeBLP2014, and vaccine strain Bartha-K61, HB98 and SA-215.
The specificity of 1.8 CPA-strip methods, sensitiveness and replica test
The genomic DNA of TJ plants of PPV, PRRSV, CSFV, PCV2 and PRV is extracted, is detected with CPA-strip methods, evaluate it
Specificity.The recombinant plasmid pMD-18T-PRV of gradient dilution detects the sensitiveness of the method.From the TJ plants of base that infection cell is extracted
Because of a group DNA, with 3 different virus titres, 5 replica tests and between group in group are carried out.
The accordance analysis of 1.9 CPA-strip and triple fluorescent quantitative PCR, virus purification
Between 2013-2016, the samples such as brain, spleen and lymph node being collected from the sick dead pig with obvious clinical symptoms
Product, are detected totally using CPA-strip by 220 parts.All samples application triple fluorescent quantitative PCR (Meng, X.Y., Luo,
Y.,Liu,Y.,Shao,L.,Sun,Y.,Li,Y.,Li,S.,Ji,S.,Qiu,H.J.,2016.A triplex real-time
PCR for differential detection of classical,variant and Bartha-K61vaccine
Strains of pseudorabies.Arch.Virol.161,2425-2430.) and virus purification verified, then
DNA sequencing is carried out to positive.
2nd, experimental result
The selection of 2.1 CPA-strip primers and probe
The present invention is combined the detection side of method (CPA-strip) for detecting the cross primer amplification-immune chromatography test paper of PRV
Method principle is shown in Fig. 2.
The selection result of primer and probe shows that scheme 1 and scheme 2, may be with pig genes when negative sample is detected
Group DNA produces cross reaction, generates trapezoid-shaped strips, it is impossible to accurately detect PRV (Fig. 3 A, B).Scheme 3 is when negative sample is detected
Without band generation, and pig genomic DNA no cross reaction, while there is obvious band generation (Fig. 3 C) when positive is detected.
The middle probe of scheme 3 and primer are compared with known PRV strains sequence in NCBI, as a result shows that probe and primer can be correct
Identification known array (Fig. 4).Therefore, be used for the probe and primer in scheme 3 in subsequent experimental by the present invention.
2.2 probe synthesis analysis
The end modified probe application mass spectral analysis for having biotin and FITC is detected.Result shows, probe PRV-3s
With PRV-1s compared with unmodified general primer, molecular weight rises to 6148.5Da, 5524Da and rises to from 5606.8Da respectively
5930.9Da (Fig. 5).Result shows that probe is successfully prepared.
The CPA reaction conditions of 2.3 optimizations
To improve the amplification efficiency and sensitiveness of CPA reactions, reaction temperature and amplification reinforcing agent need to carry out condition optimizing.Knot
Fruit shows, under identical temperature conditionss, when reaction system adds glycine betaine, the amplified production of generation is most.Meanwhile, reaction temperature
For 61 DEG C when, amplified production content is higher than amplified production content (Fig. 6) under the conditions of other reaction temperatures.Therefore, reaction temperature is
61 DEG C while be CPA optimum reaction conditions when expanding reinforcing agent for glycine betaine.This reaction condition is applied to subsequent experimental.
The specificity of 2.4 CPA-strip methods, sensitiveness and repeatability
CPA-strip methods are detected to PRV difference strains.Result shows that the method is in detection PRV
At BJKJZ2015, HeBLP2014, HLJMDJ2013, TJ, Min-A and SC plant, developed the color at detection line and nature controlling line, show the party
Method can accurately detect PRV classics strains and variant (Fig. 7).Meanwhile, the method is in detection Bartha-K61, HB98 and SA-215
During strain, developed the color only at nature controlling line, show that the method can not detect PRV vaccine strains, can be with the wild poison of specific detection PRV.CPA-
Strip methods detect to the Prevention of Common Occurrence Porcine Disease cause of disease such as CSFV, PCV2, PPV and PRRSV, as a result all feminine genders (Fig. 8).
Sensitivity analysis testing result shows, naked eyes occur when standard plasmid is 200 copies, at detection paper line can know
Other minimum colour developing change.Therefore, the lowest detection of the method is limited to 200 copies (Fig. 9).
Repeated testing result shows, without the color change that naked eyes are recognizable at same concentration detection line.
The accordance of 2.5 CPA-strip and other methods
CPA-strip methods detect, as a result show that it is the positive, 193 to have 27 sample detection results to 220 samples
Individual sample is feminine gender.Accordance analysis display, the coincidence rate of CPA-strip and triple fluorescent quantitative PCR is 100% (220/
220) it is, 98.2% (216/220) (table 2) with the coincidence rate of virus purification.Wherein 6 samples are determined PCR and are detected through triple fluorescent
Analyzed with DNA sequencing and shown, sample contains Bartha-K61 vaccine strains, but CPA-strip is detected as feminine gender.DNA sequencing point
Analysis display, in 27 positives, 23 samples contain PRV variants, and 4 samples contain PRV classics strains.Result above table
Bright, the CPA-strip methods that the present invention sets up can be used for now detecting for PRV street strains.
The CPA-strip of table 2 and triple fluorescent quantitative PCR, the coincidence rate of virus purification
SEQUENCE LISTING
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>Cross primer amplification-the immune chromatography test paper of detection pseudorabies virus street strain is combined the primer and probe of method
Group and kit
<130> HEB
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> artifical sequence
<400> 1
cccagcccaa gatgacg 17
<210> 2
<211> 17
<212> DNA
<213> artifical sequence
<400> 2
tggtctcaac cccggtg 17
<210> 3
<211> 40
<212> DNA
<213> artifical sequence
<400> 3
tgcccaggtt taaaacggtt ttgcataatt ttgtgggtgg 40
<210> 4
<211> 18
<212> DNA
<213> artifical sequence
<400> 4
gcggacggag ataaaacg 18
<210> 5
<211> 18
<212> DNA
<213> artifical sequence
<400> 5
tgcccaggtt taaaacgg 18
<210> 6
<211> 17
<212> DNA
<213> artifical sequence
<400> 6
cccagcccaa gatgacg 17
<210> 7
<211> 17
<212> DNA
<213> artifical sequence
<400> 7
tggtctcaac cccggtg 17
<210> 8
<211> 40
<212> DNA
<213> artifical sequence
<400> 8
gaacaataaa aaggtggtgt tttgcccagg tttaaaacgg 40
<210> 9
<211> 19
<212> DNA
<213> artifical sequence
<400> 9
tgcataattt tgtgggtgg 19
<210> 10
<211> 19
<212> DNA
<213> artifical sequence
<400> 10
gaacaataaa aaggtggtg 19
<210> 11
<211> 17
<212> DNA
<213> artifical sequence
<400> 11
cccagcccaa gatgacg 17
<210> 12
<211> 17
<212> DNA
<213> artifical sequence
<400> 12
tggtctcaac cccggtg 17
<210> 13
<211> 40
<212> DNA
<213> artifical sequence
<400> 13
tgcccaggtt taaaacggtt tgaacaataa aaaggtggtg 40
<210> 14
<211> 18
<212> DNA
<213> artifical sequence
<400> 14
gcggacggag ataaaacg 18
<210> 15
<211> 18
<212> DNA
<213> artifical sequence
<400> 15
tgcccaggtt taaaacgg 18
Claims (10)
1. it is used to detect that the cross primer amplification-immune chromatography test paper of pseudorabies virus street strain is combined the primer and probe of method
Group, including two Outside primers, a cross primer and two probes；Characterized in that, being selected from following three groups of primers and probe
In any one group：
(1) nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.1, and the nucleotides sequence of the Outside primer 2 is classified as
Shown in SEQ ID No.2；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.3；The nucleotides sequence of the probe 1
It is classified as shown in SEQ ID No.4, the nucleotides sequence of the probe 2 is classified as shown in SEQ ID No.5；
(2) nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.6, and the nucleotides sequence of the Outside primer 2 is classified as
Shown in SEQ ID No.7；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.8；The nucleotides sequence of the probe 1
It is classified as shown in SEQ ID No.9, the nucleotides sequence of the probe 2 is classified as shown in SEQ ID No.10；
(3) nucleotides sequence of the Outside primer 1 is classified as shown in SEQ ID No.11, the nucleotide sequence of the Outside primer 2
Shown in SEQ ID No.12；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.13；The nucleosides of the probe 1
Acid sequence is that the nucleotides sequence of the probe 2 is classified as shown in SEQ ID No.15 shown in SEQ ID No.14.
2. application of the primer and probe groups described in claim 1 in detection pseudorabies virus street strain.
3. a kind of cross primer amplification-immune chromatography test paper for detecting pseudorabies virus street strain is combined kit, including：Hand over
Fork primer amplifing reagent and immuno-chromatographic test paper strip；Wherein, the cross primer amplifing reagent includes：Outside primer 1, outside draws
Thing 2, cross primer, probe 1, probe 2, dNTP, Mg2+, 10 × ThermoPol buffer solutions, Bst archaeal dna polymerases and amplification enhancing
Agent；It is characterized in that：The Outside primer 1, Outside primer 2, cross primer, probe 1 and probe 2 are selected from described in claim 1
Primer and probe groups；The 5 ' of the probe 1-end is connected with FITC, and 5 '-end of the probe 2 is connected with biotin.
4. kit is combined according to the cross primer amplification-immune chromatography test paper described in claim 3, it is characterised in that：It is described
The nucleotides sequence of Outside primer 1 is classified as shown in SEQ ID No.1, and the nucleotides sequence of the Outside primer 2 is classified as SEQ ID No.2
It is shown；The nucleotides sequence of the cross primer is classified as shown in SEQ ID No.3；The nucleotides sequence of the probe 1 is classified as SEQ ID
Shown in No.4, the nucleotides sequence of the probe 2 is classified as shown in SEQ ID No.5.
5. kit is combined according to the cross primer amplification-immune chromatography test paper described in claim 3, it is characterised in that：It is described
Amplification reinforcing agent is selected from glycine betaine or dimethyl sulfoxide (DMSO), preferably glycine betaine.
6. kit is combined according to the cross primer amplification-immune chromatography test paper described in claim 3, it is characterised in that utilize
The CPA reaction systems that the cross primer amplifing reagent is set up include：The cumulative volume of reaction system is 25.0 μ L；Wherein, 10 μM
The μ L of cross primer 2.0,10 μM of the μ L of Outside primer 1 0.5,10 μM of the μ L of Outside primer 2 0.5,10 μM of the μ L of probe 1 1.0,10 μM of spies
The μ L of pin 2 1.0, the μ L of 10mM dNTP 2.5,100mM Mg2+1.0 μ L, the μ L of 10 × ThermoPol buffer solutions 2.5, the Bst of 8U/ μ L
The μ L of archaeal dna polymerase 1.0, the μ L of genomic DNA 2.0, the amplification μ L of reinforcing agent 2.5, with deionized water polishing to 25.0 μ L.
The program expanded using the cross primer amplifing reagent is：59 DEG C of -67 DEG C of constant-temperature amplification 60min；Preferably, 61
DEG C constant-temperature amplification 60min.
7. kit is combined according to the cross primer amplification-immune chromatography test paper described in claim 3, it is characterised in that described
Immuno-chromatographic test paper strip includes：Sample pad, pad, detection line, nature controlling line, adsorptive pads and PVC board；
Wherein, antibiotin monoclonal antibody is coated with the detection line, mountain sheep anti-mouse igg is coated with the nature controlling line,
Contain the gold nano grain of pan coating anti-FITC monoclonal antibody in the pad.
8. the cross primer amplification-immune chromatography test paper combination kit described in claim 3 to 7 any one is pseudo- mad in detection
Application in dog disease field virus.
9. a kind of cross primer amplification-immune chromatography test paper for detecting pseudorabies virus street strain is combined method, it is characterised in that
Comprise the following steps：
(1) virus genom DNA is extracted from detected sample；(2) it is template with the DNA for extracting, with described in claim 1
Primer and probe groups are set up CPA reaction systems and are expanded；(3) by amplified production loading immuno-chromatographic test paper strip, if exempted from
Developed the color at the detection line and nature controlling line of epidemic disease chromatograph test strip, then result judgement is the positive；If developed the color only at nature controlling line,
Result judgement is feminine gender.
10. method is combined according to the cross primer amplification-immune chromatography test paper described in claim 9, it is characterised in that the CPA
Reaction system includes：The cumulative volume of reaction system is 25.0 μ L；Wherein, the 10 μM of μ L of cross primer 2.0,10 μM of Outside primers 1
0.5 μ L, 10 μM of μ L of Outside primer 2 0.5,10 μM of μ L of probe 1 1.0,10 μM of μ L of probe 2 1.0, the μ L of 10mM dNTP 2.5,
100mM Mg2+1.0 μ L, the μ L of 10 × ThermoPol buffer solutions 2.5, the μ L of Bst archaeal dna polymerases 1.0 of 8U/ μ L, genomic DNA
2.0 μ L, the amplification μ L of reinforcing agent 2.5, with deionized water polishing to 25.0 μ L.
The program of the amplification is：59 DEG C of -67 DEG C of constant-temperature amplification 60min；Preferably, 61 DEG C of constant-temperature amplification 60min；
The 5 ' of the probe 1-end is connected with FITC, and 5 '-end of the probe 2 is connected with biotin；The amplification reinforcing agent choosing
From glycine betaine or dimethyl sulfoxide (DMSO), preferably glycine betaine；
The immuno-chromatographic test paper strip includes：Sample pad, pad, detection line, nature controlling line, adsorptive pads and PVC board；Wherein, institute
State and be coated with detection line antibiotin monoclonal antibody, mountain sheep anti-mouse igg is coated with the nature controlling line, in the pad
Gold nano grain containing pan coating anti-FITC monoclonal antibody.
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| CN110016513A (en) * | 2019-03-31 | 2019-07-16 | 中国农业科学院兰州兽医研究所 | A kind of horse Taylor worm identification detection kit |
| CN112725530A (en) * | 2021-01-19 | 2021-04-30 | 中国计量大学 | Cross primer amplification primer group for detecting bean pod mottle virus, kit and application |
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| CN112725530A (en) * | 2021-01-19 | 2021-04-30 | 中国计量大学 | Cross primer amplification primer group for detecting bean pod mottle virus, kit and application |
| CN113136456A (en) * | 2021-04-25 | 2021-07-20 | 武汉科前生物股份有限公司 | Fluorescent quantitative PCR detection kit for identifying porcine pseudorabies virus gene deletion vaccine strain and wild strain |
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Application publication date: 20170613 |