CN107236825A - For real time RPA quick detections and the nucleic acid and method of the wild poison of differentiation PRV and vaccine virus - Google Patents
For real time RPA quick detections and the nucleic acid and method of the wild poison of differentiation PRV and vaccine virus Download PDFInfo
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Abstract
The invention belongs to biological technical field, a kind of nucleic acid and method that the wild poison of PRV and vaccine virus are detected for real time RPA quick discriminatings is specifically disclosed.According to the conservative region design specificity RPA primers and exo probes of gB and gE genes in PRV genomes.The wild poison of real time RPA quick discriminatings detection PRV of offer and the method for vaccine virus can be at 39 DEG C in 20min, specific detection and the differentiation to the wild poison of PRV and vaccine virus are realized in a reaction system, and the swine disease poison common with other is without cross reaction, its quick, easy to operate, reliable results of reaction, be highly suitable for veterinary diagnostic laboratory and plant scene PRV detection, especially scarcity of resources backwoodsman plant Site Detection.
Description
Technical field
The invention belongs to biological technical field, and in particular to wild for real-time RPA quick detections and differentiation PRV
The nucleic acid and method of poison and vaccine virus.
Background technology
Pseudo- mad dog (Pseudorabies, PR), also referred to as AujeszkyShi diseases, are by Pseudorabies virus
A kind of important epidemic disease for betiding pig and other animals, can cause foster caused by (Pseudorabies virus, PRV)
The serious economic loss of pig industry.PRV, belongs to herpetoviridae, Varicellavirus is a kind of double-stranded DNA disease with cyst membrane
Poison.Pig is PRV natural reservoir (of bird flu viruses), is also that unique PRV that is resistant to infects, forms the animal of latent infection.PRV can infect
Simultaneously different clinical symptoms are presented in the pig of different growth phases：Nervous symptoms and high mortality are mainly shown as in newborn piglet；
Respiratory symptom is mainly shown as in Adult Pig；Breeding difficulty is mainly shown as in farrowing sow.In addition to pig, PRV is also
Can infect a variety of different mammals, including ruminant, Canidae and rodent, infect it is latter as show after it is dead
Die.
In China, the especially small-sized and medium-sized pig farm in pig farm is very low to the attention degree of bio-safety, and this is also resulted in
The control and removing of pseudo- mad dog are more difficult.On many farms, not to infection PRV or die from PRV infection pig carry out it is buried
Or burning disposal, but various forms of sale have been carried out, this also results in the wide-scale distribution of the pseudo- mad dog of China.
Bartha-K61 strain virus has lacked gE genes, is the pseudo- mad dog attenuated vaccine strain being most widely used at present.I
All pseudo- mad dog attenuated vaccines that state uses at present are Bartha-K61 plants.The use of pseudo- mad dog attenuated vaccine can be effective
The pseudo- mad dog epidemic situation of control, substantially reduce the morbidity and mortality of newborn piglet, but using pig can not be removed after vaccine
The wild poison of unborn PRV, can not prevent follow-up wild virus infection in vivo.In infected pigs' body, PRV can be formed all the life
Latent infection, and once activate, it becomes possible to cause the wide-scale distribution of the wild poison of PRV.Since year ends 2011, one kind has
The PRV variants of more highly pathogenicity, which were immunized in the multiple areas of China in the pig farm of Bartha-K61 vaccines, to be occurred, and is caused
Serious economic loss.Therefore, a kind of simple, quickly detection and differentiation PRV street strains and vaccine virus method is set up,
Have very important significance.
A variety of PRV detections based on DNA cloning technology and discrimination method, such as real time fluorescent PCR method are had built up at present
(real-time PCR), loop-mediated isothermal amplification method (LAMP) and nano particle PCR (nanoPCR) method.But, it is above-mentioned
There is certain deficiency in method, it is impossible to be effectively applied to production line and Site Detection.Real-time PCR methods are needed
Will the expensive equipment of specialty and special technical staff's operation；The result judgement of LAMP method and nanoPCR methods needs to carry out
Agarose gel electrophoresis, greatly reduces detection efficiency, and false positive rate is higher, as a result unreliable.
Recombinase polymeric enzymatic amplification (RPA) is a kind of isothermal DNA amplification technology, recombinase combination primer formation albumen-
DNA mixtures simultaneously start the homologous sequence found on template DNA.After homologous sequence positioning, then it can trigger strand replacement reaction, draw
Thing is attached in corresponding templates, polymerase and then the startup DNA synthesis since the end of primer 3 '.It is the same with PCR, two primers
The level several levels amplification to target gene can be started.Need to add an exo in real-time fluorescence (real-time) RPA reaction systems
The fluorescence signal produced in probe, amplification procedure realizes real-time detection by fluorescence detector.But at present due to primer and exo
The design difficulty of probe is larger, is typically relatively inaccessible to preferable Detection results.
The content of the invention
It is an object of the invention to provide the nucleic acid that one group is used for real-time RPA quick detections PRV viruses, this hair
Another bright purpose is that providing one group is used for the core that real-time RPA quick discriminatings detect the wild poison of PRV and vaccine virus
Acid.It is used for the wild poison of real-time RPA quick discriminatings detection PRV and vaccine virus it is also another object of the present invention to provide one kind
Nucleic acid kit and its detection method.
To achieve the above object, the present invention use following technical scheme for：
One group is used for the nucleic acid of real-time RPA quick detections PRV viruses, including for detecting PRV sense primer
1st, anti-sense primer 1 and exo probes 1；Wherein, the sequence of the sense primer 1 is as shown in SEQ ID No.1, the anti-sense primer
1 sequence as shown in SEQ ID No.2, the sequence of the exo probes 1 as shown in SEQ ID No.3, wherein, the exo is visited
31st T kilobase marker fluorophor of pin 1, the 32nd T kilobase marker fluorescent quenching group, the 31st T base and institute
State and tetrahydrofuran molecule is provided between the 32nd T base, 3 ' ends of the exo probes 1 are marked with C3, phosphate group, biology
Element-TEG or amino.
Nucleic acid as described above, it is preferable that it also includes the positive amplification Product Sequence 1 of PRV Viral diagnosis, the sun
Property amplified production sequence 1 include the sequence as shown in SEQ ID No.4.
One group is used for the nucleic acid that real-time RPA quick discriminatings detect the wild poison of PRV and vaccine virus, including as described above
Nucleic acid and sense primer 2, anti-sense primer 2 and exo probes 2 for detecting the wild poison of PRV, the sequence of the sense primer 2 is such as
Shown in SEQ ID No.5, the sequence of the anti-sense primer 2 is as shown in SEQ ID No.6, the sequence such as SEQ of the exo probes 2
Shown in ID No.7, the 31st T kilobase marker of the exo probes 2 has the fluorophor different from the exo probes 1, the 32nd
Position T kilobase marker fluorescent quenching groups, provided with tetrahydrofuran point between the 31st T base and the 32nd T base
Son, 3 ' ends of the exo probes 2 are marked with C3, phosphate group, biotin-TEG or amino.
Nucleic acid as described above, it is preferable that this group of nucleic acid also includes the positive amplification Product Sequence 2 of the wild poison detections of PRV,
The positive amplification Product Sequence 2 includes the sequence as shown in SEQ ID No.8.
Nucleic acid as described above, it is preferable that the fluorophor is FAM, ROX or TAMARA, the fluorescent quenching group
For BHQ1 or BHQ2.
It is a kind of to be used for the detection kit that real-time RPA quick discriminatings detect the wild poison of PRV and vaccine virus, its
Contain nucleic acid as described above.
Kit as described above, it is preferable that the kit also includes：It is recA recombinases, Bsu DNA polymerases, single-stranded
Associated proteins and exo exonucleases, reaction Buffer, magnesium acetate solution.
A kind of method that the wild poison of PRV and vaccine virus are detected for real-time RPA quick discriminatings, this method is non-examines
The detection method of disconnected purpose, specifically includes following steps：
(1) DNA of detection sample is prepared；
(2) DNA described to step (1) carries out isothermal duplication；Wherein, in reaction system, using in such as claim 3
Described nucleic acid, is placed in 39 DEG C of isothermal reactions；
(3) 20min is carried out in the isothermal reaction, while collecting fluorescence signal；
(4) result judgement：
If the fluorophor that exo probes described in testing sample 1 is marked has obvious amplification curve, while the exo probes
If the fluorophor of 2 marks has obvious amplification curve, that is, it is judged as that the wild poison of PRV is positive；
If the fluorophor that exo probes described in testing sample 1 is marked has obvious amplification curve, if while the exo is visited
The fluorophor that pin 2 is marked is judged as that PRV vaccine virus is positive without obvious amplification curve；
If the fluorophor that exo probes described in testing sample 1 is marked is without amplification curve, it is judged as that PRV viruses are cloudy
Property.
Method as described above, it is preferable that in the reaction system in the step (2), containing the sense primer 1, downstream
The final concentration of 200nmol/L of primer 1, the final concentration of 60nmol/L of the exo probes 1, the sense primer 2, downstream are drawn
The final concentration of 600nmol/L of thing 2, the final concentration of 160nmol/L of the exo probes 2.
Method as described above, it is preferable that in the step (2), while setting nuclease-free water to be negative control；Adopt
Positive control is used as with comprising the sequence as shown in SEQ ID No.4 and SEQ ID No.8.
In detection process, while negative control and positive control are provided with, for monitoring in detection process whether have dirt
Dye or operation whether specification, and whether agents useful for same effective, can fully ensure that the accurate, reliable of testing result, is prevented effectively from vacation
The generation of positive and false negative result.
Beneficial effects of the present invention are：
The invention provides the nucleic acid that can be used in the wild poison of real-time RPA quick discriminatings detection PRV and vaccine virus and
Method.Wherein, the dual real-time RPA methods of the wild poison of quick discriminating detection PRV and vaccine virus realize the inspection to PRV
Survey and differentiation is wild poison or vaccine virus, completed in same reaction system.Further analysis shows, the method provided
The classical strains of PRV and variant can be detected simultaneously.The method provided is verified using clinical sample, as a result shown,
The detection uniformity of real-time RPA and real-time PCR methods is 100%, but the inventive method (7-12min)
It is significantly faster than that real-time PCR (28-38min).The dual real-time RPA methods that the present invention is provided are pseudo- for China
The control of mad dog is significant, and all pseudo- rabies vaccines that especially current China uses are gE missings, and are retained
GB genes.
The present invention provides real-timeRPA detection method prior arts and compared, and also has the advantage that first, RPA exists
Detection is completed in 20min, and real-time PCR completion detections need about 55min, LAMP methods need 60min, and
NanoPCR then needs 90min.Secondth, RPA primer and probes are resistant to 5-9 base mispairing without influenceing testing result,
And the mispairing of probe base can cause the reduction of real-time PCR detection sensitivities, the 3rd, dual real-time RPA inspection
Real-time detection can be realized by two passages by surveying result, and interpretation of result is simple, can directly know result, and LAMP method and
NanoPCR methods then need to carry out interpretation of result into row agarose gel electrophoresis, cumbersome, as a result need wait at least more
It could be obtained after 20min.Fluorescence detector Genie III, Genie III workable for the inventive method only weigh 1.75kg, can
Rechargeable battery being capable of continuous firing one day so that differentiate that detection is possibly realized to PRV scene, this is for positioned at remote districts
Farm is particularly important.
Brief description of the drawings
Fig. 1 is sensitive amplification curve map of the method for optimizing in the present invention to gB genes.
Fig. 2 is the sensitive amplification curve map of a method for optimizing of the invention to gE genes.
Fig. 3 is specific amplification curve figure of the method for optimizing in the present invention to gB genes.
Fig. 4 is the specific amplification curve figure of a method for optimizing of the invention to gE genes.
Fig. 5 is real-time RPA methods and fluorescence PCR method of the invention to gB genetic test results contrast figures.
Fig. 6 is real-timeRPA methods and fluorescence PCR method of the invention to gE genetic test results contrast figures.
Embodiment
Illustrate present invention with reference to specific embodiment, but should not be construed as limiting the invention.Not
In the case of spirit of the invention and essence, the modifications or substitutions made to the inventive method, step or condition are belonged to
The scope of the present invention.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The primer of embodiment 1, the design of probe
In the present invention, from Pseudorabies virus gB genes as detection target gene, because gB genes have it is highly conserved
And exist in all wild poison and vaccine virus, so being used as detection rabies viruses from gB genes；In all pseudo- rabies vaccine poison
GE genes are lacked, so it is vaccine virus or wild poison to be used to distinguish gE genes using detection gE genes.
Using recombinase polymeric enzymatic amplification technology (RPA), carry out the detection and discriminating to Pseudorabies virus, primer pair and
The present invention of exo probes is successfully crucial, and the primer and probe of the technology are designed without special software progress, Zhi Nengyi
Manually design, and the primer combination of probe for needing design different are verified, so as to ensure the specific and sensitive of detection
Property.In RPA design of primers, (1) should avoid before the end of primer 5 3-5 base from being continuous G, and to be preferably C (or be at 5 ends
Pyrimidine).Last 3 bases in the end of primer 3 are preferably G and C.(2) try one's best and avoid occurring special sequence in primer, as repeated
Long string of a certain base.(3) G/C content is moderate, it is impossible to too high (>70%) or it is too low (<30%).(4) in primer or between primer
With correspondence as far as possible avoid, prevent the generation of primer dimer.(5) try one's best and avoid the formation of primer secondary structure, prevent primer
Between interaction, avoid the formation of hairpin structure.Exo probes are one section of homologous nucleotides of same target extension increasing sequence, wherein de- comprising one
Base nucleosides acid mimic (THF), its both sides are respectively dT- fluorophors and dT- fluorescent quenching groups.Meanwhile, the end of probe 3
It is closed by appropriate modification (C3-spacer, phosphate group, biotin-TEG or amino), so as to prevent possible polymerization
Enzymatic amplification and extension.At present, the inner marker of exo probes can only occur in T bases, and two in probe dT is apart
No more than 6 (1-5) bases.In the present invention, PRV sequences all in current Genbank are compared,
The high conservative region design primed probe of gB and gE genes, can distinguish the wild poison of PRV and vaccine virus in same reaction system,
No cross reaction had both been required between primer, exo probes, the specificity of primed probe amplification, design are fully ensured that again
Multipair primer combination of probe has been synthesized, has fully been verified, the optimal primer combination of probe of screening expanding effect.
Specifically, according to GQ325658.1 in Genbank, KT948054.1, KP009898.1, KT948053.1,
KT818618.1, KP009897.1, KT948054.1, KP710982.1, KJ526438.1, retrieve the gB bases of Pseudorabies virus
Because of fragment, design upstream and downstream primer 1 and exo probes 1.Sense primer 1, the downstream of Pseudorabies virus are detected for constant-temperature amplification
Primer 1 (RPA-gB-F, RPA-gB-R) and exo probes 1 (RPA-gB-P), it is specific as shown in table 1.
Exo probes contain a base analogies tetrahydrofuran molecule (tetrahydrofuran, THF), T HF molecules two
Side is respectively provided with fluorophor and fluorescent quenching group, and probe 3' ends carry the blocker for preventing that probe from extending.When probe and target
DNA is combined to form after double stranded heteroduplex DNA structure, and exo III is as a kind of DNA repair enzymes, and recognizing THF sites and cutting probe makes
Fluorophor and fluorescent quenching group separation are so as to produce fluorescence, two thymidines in the centre position of exo probes 1
THF is designed between one fluorophor of mark and a fluorescent quenching group respectively on acid, two groups, the site can quilt
With 3'-5' 5 prime excision enzyme activities exonuclease III identification, cutting, dissociate fluorophor so that send fluorescence signal with
Captured afterwards by the instrument of fluoroscopic examination.By substantial amounts of experimental verification, as a result show, present invention selection is the of exo probes 1
31 T kilobase marker fluorophors, the 32nd T kilobase marker fluorescent quenching group, the 31st T base and the 32nd T base it
Between be provided with THF, 3 ' end connection C3, phosphate group, biotin-TEG or the amino of exo probes 1, Detection results are optimal, are marked
Fluorophor be FAM when, corresponding quenching group be BHQ1；When the fluorophor of mark is TAMARA or ROX, correspondence
Quenching group be BHQ2；3 ' end connection C3, phosphate group, biotin-TEG or the amino of probe, empirical tests, using above-mentioned
When group is detected, Detection results are identical.Used when exo probes 1 are specifically synthesized in the present embodiment
TCTACTACAAGAACGTCATCGTCACGACCG(FAM-dT)(THF)(BHQ1-dT) -GGTCCGGGAGCACGTA-C3。
The product of RPA-gB-F and RPA-gB-R amplifications is 333bp, and particular sequence is as shown in SEQ ID No. 4：
GCTCTTCAAGGAGAACATCGCCCCGCACAAGTTCAAGGCCCA
CATCTACTACAAGAACGTCATCGTCACGACCGTGTGGTCCGGGAGCAC
GTACGCGGCCATCACGAACCGCTTCACGGACCGCGTGCCCGTCCCCGT
GCAGGAGATCACGGACGTGATCGACCGCCGCGGCAAGTGCGTCTCCA
AGGCCGAGTACGTGCGCAACAACCACAAGGTGACCGCCTTCGACCGC
GACGAGAACCCCGTCGAGGTGGACCTGCGCCCCTCGCGCCTGAACGC
GCTCGGCACCCGCGGCTGGCACACCACCAACGACACCTACACCAAGA TCGGCGC.It can use and contain SEQ ID No.4
Sequence used as positive control.
On the basis of the good primer that PRV viruses are detected for real-time RPA of design optimization, exo probes, if
Count the nucleic acid for distinguishing the wild poison of PRV and vaccine virus, from gE genes (AF207700.1, KF 017615.1, KM189913.1,
KM983048.1, KF360835.1, KJ789182.1, KU057 086.1, AY170318.1), sense primer 2, downstream after optimization
The sequence of primer 2 (RPA-gE-F, RPA-g E-R) and exo probes 2 (RPA-gE-P), as shown in table 1.Wherein, specifically exist
Exo probes it is 2-in-1 into when be that the 31st and the 32nd centre are provided with THF, two T kilobase markers difference mark fluorescent on both sides
Group and fluorescent quenching group, fluorophor is using ROX and fluorescent quenching group using B HQ2 during synthesis, and 3 ' ends connect C3.I.e.
Specially CCGAGGAGGCGCCCCGCTCCGGCTTCGAC G (ROX-dT) (THF) (BHQ2-dT) GGTTCCGCGATCCGGA-
C3。
Sense primer 2, the size of the amplified production of anti-sense primer 2 are 156bp, and its sequence is as shown in SEQ ID No.8：
ACCCCGAGGACGAGTTCAGCAGCGACGAGGACGACGG
GCTGTACGTGCGCCCCGAGGAGGCGCCCCGCTCCGGCTTCGACGTCTG
GTTCCGCGATCCGGAGAAGCCGGAAGTGACGAATGGACCCAACTATG GCGTGACCGCCAACCGCCTGTTGA。
Primer and probe sequence is shown in Table 1, and all primer and probes are synthesized by Shanghai life work.
The primer probe sequence information of table 1
Wherein, R represents A or G, Y represent C or T.The dual real-time fluorescence RPA methods of embodiment 2
The method that the wild poison of PRV and vaccine virus are detected for real-time RPA quick discriminatings, i.e. dual real-time fluorescence RPA
The wild poison of method quick discriminating detection PRV and vaccine virus, specifically include following steps：(1) DNA of detection sample is prepared；
(2) isothermal duplication is carried out to DNA sample in step (1)：Use the TwistAm pTM exo kit of Twist companies
(TwistDX, Cambridge, UK), reaction system uses 50 μ L.In 50 μ L reaction systems, for the primer of gB genes
(RPA-gB-F/RPA-gB-R) and gE genes primer (RP A-gE-F/RPA-gE-R) concentration can be respectively adopted 200nM,
400nM, 500nM and 600nM, the exo probes (RPA-gE-P) of exo probes (RPA-gB-P) and gE genes for gB genes
Concentration and probe concentration is respectively 60nM, 120nM, 160nM and 200nM.Also include 29.5 μ L e xo Buffer, 2.5 in reaction system
μ L concentration is 280mM magnesium acetate, 1 μ L viral DNAs and 11.9 μ L ddH2O.By all reagents outside template and magnesium acetate
It is transferred to after premix containing lyophilized enzyme preparation (recA recombinases, Bsu archaeal dna polymerases, single strand binding protein and exo exonucleases
Enzyme) 0.2mL reaction tubes in, it is and full and uniform.1 μ L templates are added in reaction tube, and 2.5 μ L magnesium acetates are added in reaction
In lid, cover tightly rear brief centrifugation and be vortexed, be put into Genie III, 39 DEG C of isothermal reactions；
(3) 20min is carried out in 39 DEG C of isothermal reactions, while collecting fluorescence signal：Using Channel-1 passages (Blue,
Excitation wavelength is 470nm, and Detection wavelength is 510-560nm) collect gB gene magnifications fluorescence signal；It is logical using Channel-2
Collect the fluorescence signal of gE gene magnifications in road (Yellow, excitation wavelength 590nm, Detection wavelength are more than 62 0nm).
(4) result judgement：If the fluorophor that exo probes described in testing sample 1 is marked has obvious amplification curve, together
If the fluorophor that Shi Suoshu exo probes 2 are marked has obvious amplification curve, that is, it is judged as that the wild poison of PRV is positive, if simultaneously described
The fluorophor that exo probes 2 are marked is judged as that PR V vaccine virus is positive without obvious amplification curve；If exo described in testing sample
The fluorophor that probe 1 is marked then is judged as that PRV viruses are negative without amplification curve.
Preferably, when the primer concentration of gB genes is 200nM, concentration and probe concentration is 60nM, and the primer concentration of gE genes is
600nM, when concentration and probe concentration is 160nM, Channel-1 (gB genes) and Channe l-2 (gE genes) passage can detect glimmering
Optical signal, and good amplification curve is presented.
In order to avoid agents useful for same fails or is contaminated in the method that the present invention is set up, it is right as the positive to be additionally provided with
According to and negative control reagent, negative control uses nuclease-free water；
Positive control is contained such as SEQ ID using the DNA for carrying amplified production, the nucleotide sequence of amplified production
Sequence shown in No.4 and SEQ ID No.8.
The design of negative control can effectively verify whether agents useful for same is contaminated, it is to avoid false positive occurs, positive control
Design can effectively verify the validity of agents useful for same, it is to avoid the generation of false negative.
The detection of the sensitivity of embodiment 3
The preparation of 1.DNA standard items
Using PRV Fa strain virus genomic DNAs template, with pRVgB-F/pRVgB-R a nd pRVgE-F/ in table 1
PRVgE-R is primer, and performing PCR amplification is entered respectively, gB genes and gE full length genes is obtained.After PCR primer is reclaimed, connection
PMD19-T carriers, construction recombination plasmid pRV-gB and pRV-gE, and it is transformed into competent cell DH5 α.Will the correct sun of sequencing
Property clone's incubated overnight after extract plasmid and determine concentration using ND 2000c.According to formula：Copy number (copies/ μ L)=
6.02×1023× concentration (ng/ μ L) × 10-9/ (base number × 660), calculate recombinant plasmid copy number.By recombinant plasmid pRV-
GB and pRV-gE carries out 10 times of doubling dilutions, makes in the range of 106-100Copy/μ L, as DNA standard items, is stored in -80 DEG C
It is standby.
2. using the dual real-time fluorescence RPA method sensitivity techniques in embodiment 2
The DNA standard items that 1 μ L are serially diluted are taken as template, using preferably method enters line sensitivity point in embodiment 2
Analysis, wherein, the primer concentration of gB genes is 200nM in reaction solution, and concentration and probe concentration is 60nM, and the primer concentration of gE genes is
600nM, concentration and probe concentration is 160nM.
Using concentration as 106To 100Copies gB and gE DNA standard items carry out sensitivity technique, each as template
Concentration carries out 8 real-time RPA detections.As a result show, when gB and gE standard concentrations are 106To 102During copies, gB
It is the positive with 8 detections of gE genes；Its testing result, Channel-1 (gB genes) amplification curve as shown in figure 1,
Channel-2 (gE genes) amplification curve is as shown in Figure 2.When gB and gE standard concentrations are 101During copies, gB genes 4
Secondary test positive, and 3 test positive of gE genes；When gB and gE standard concentrations are 100During copies, gB and gE bases
Because 8 detections are feminine gender.As a result illustrate, for gB and gE genes, detection sensitivity of the invention can reach
101copies。
The specific detection of embodiment 4
Using preferably dual real-time fluorescence RPA methods in embodiment 2, to being stored in Hebei Entry-Exit Inspection and Quarantine Bureau
The Pseudorabies virus (RPV, Fa, SH151218 plant) in inspection and quarantine technique center laboratory, porcine reproductive and respiratory syndrome virus
(PRRSV, strain HB-Xl), encephalomyocarditis virus (EMCV, strain BD) and porcine circovirus 2 type (PCV-2,
strain HB-MC1)；Wherein 51218 plants of PRV SH1 are the change on the Hebei pig farm for being isolated from being immunized Bartha-K61 vaccines
Different strain.
And from Chinese commodity attenuated vaccine：Pseudorabies virus (Bartha-K61), CSFV (C SFV, strain
AV1412), pig parvoviral (PPV, strain BJ-2), this 8 kinds viruses are detected.
Using these virus genom DNAs or cDNA as template, double fluorescent RPA reaction condition is：39 DEG C,
20min。
Result is PRV virus vaccine strains Bartha-K61 only gB gene masculines (A), and PRV street strains Fa and SH151218
Then gB and gE are positive (B).Amplification curve as shown in Figure 3 and Figure 4, wherein, 1：Pseudorabies virus (Fa)；2：Pseudoabies
Malicious (SH151218)；3：Pseudorabies virus (Bartha-K61) 4：CSFV；5：Porcine reproductive and respiratory syndrome virus；6：
Porcine encephalomyocarditis virus；7：Pig parvoviral；8：Porcine circovirus 2 type.
As a result show, the double fluorescent RPA that the present invention is provided specific can be detected and be distinguished PRV vaccine strains and open country
Strain.
Detection of the embodiment 5 to Strain and clinical sample
1. the source of Strain and clinical sample
Pseudorabies virus (RPV, Fa plants, SH151218 plants and NQY160308 plants) is stored in Hebei inspection and quarantining for import/export
Office inspection and quarantine technique center laboratory.SH151218 plants and NQY160308 plants of PRV is to be isolated from that Bartha- was immunized
The variant on the Hebei pig farm of K61 vaccines.Bartha-K61 plants of Chinese commodity attenuated vaccine of PRV
37 parts of clinical samples, include brain, lymph node, lung, kidney and the whole blood of 9 parts of dead piglets, the brain of 8 parts of dead recoon dogs,
Lymph node, lung, kidney, and 20 parts of brains and lymph node from healthy slaughter pig, are all from Hebei Province plant.For group
Tissue samples, take after 10mg abundant homogenizeds in the sterile PBS of 1mL, 2000rpm centrifugations 10min, take supernatant to be used for viral DNA
Extract.For whole blood sample, 1mL is taken to be extracted for viral DNA.
2. viral DNA/RNA extractions and RNA reverse transcription
Use TIANamp Virus genomic DNA/RNA kit (TIANGEN Biotech (Beijing) Co., Ltd., north
Capital) carry out viral nucleic acid extraction.100ng viral RNAs are taken, Primescript II 1st strand cDNA are used
Synthesis kit (precious bioengineering (Dalian) Co., Ltd, Dalian) carry out transcription and obtain cDNA.Use ND-2000c cores
Measuring acid concentration instrument (Wilmington, USA) carries out viral DNA, cDNA concentration mensurations.All viral DNAs and cDNA are protected
It is stored in -80 DEG C.
3. the checking pair dual real-time RPA methods
Using preferably dual real-time RPA methods in embodiment 2 to the wild poison of 3 plants of PRV (Fa, SH151218 and
NQY160308), 1 strain vaccine is malicious (Bartha-K61), and 37 parts of clinical samples carry out PRV discriminating detection, and will detection
As a result with document W.Ma, K.M.Lager, J.A.Richt, W.C.Stoffregen, F.Zhou, K.J.Yoon,
Development of real-time polymerase chain reaction assays for rapid detection
and differentiation of wild-type pseudorabies and gene-deleted vaccine
Real-time PCR methods in viruses, J Vet Diagn Invest, 20 (2008) 440-447. are compared, its
The sequence of primer and probe used in middle real-time PCR is shown in Table 1, and using Premix Ex Taq, (precious bioengineering is (big
Even) Co., Ltd, Dalian) the middle QuantiTect probe PCR Master Mix used are offered instead of original text, use
ABI7500 carries out PRV real-time PCR detections.
4. result
The real-time RPA and existing real-time PCR that the present invention is provided are one to the detection knot of all samples
(21 parts of positives, the 20 parts of feminine genders) caused, wherein, 21 parts of positive testing results are shown in Table 2.Further analysis shows, two methods
Detection uniformity be 100%.All sample P RV gB and gE genes from piglet and recoon dog are positive (being shown in Table 2), and
Sample P RV gB and the gE genes of health pig are feminine gender.All PRV Strain (Fa, SH151218, NQY160308 and
Bartha-K61) it is gB gene masculines, and only Bartha-K61 is gE gene negatives (being shown in Table 2).For positive clinical sample
Product, real-time RPA detection time is 7-12min, and the time required for real-time PCR is about 28-38min,
The quick of real-time RPA methods is absolutely proved.
With the testing result (TT) of real-time RPA methods for ordinate, with the detection of real-time PCR methods
As a result (Ct) is abscissa, and the linear regression analysis carried out using Prism softwares is shown, inspection of the two methods to gB genes (A)
Survey correlation (R2) it is 0.983, to the detection correlation (R of gE genes (B)2) it is 0.992, as a result see Fig. 5 and Fig. 6.
The double fluorescent RPA of table 2 and fluorescent PCR compare different PRV and clinical sample testing result
All clinical samples from dead piglet and recoon dog are that the wild poison of PRV is positive in the present embodiment, subsequently through
PRV separating resultings also demonstrate that the PRV infection of piglet and recoon dog.Further investigation shows, recoon dog is eating the piglet of death
Afterwards, pseudo- mad dog clinical symptoms are occurred in that and dead.
The present invention, which is provided, can differentiate the dual real-time RPA methods of the wild poison of detection PRV and vaccine virus simultaneously.Should
The portability of method can be effectively applied to the Site Detection of pseudo- mad dog, especially scarcity of resources area, while to puppet
Epidemiology survey, prevention and control and the removing of mad dog are also significant.
SEQUENCE LISTING
<110>Technology Center Of Hebei Import and Export Inspection and Quarantine Bureau, the gull Venture Capital bio tech ltd of Beijing one hundred
<120>For real-time RPA quick detections and the nucleic acid and method of the wild poison of differentiation PRV and vaccine virus
<130>
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213>It is artificial synthesized
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gctcttcaag gagaacatcg ccccgcacaa 30
<210> 2
<211> 31
<212> DNA
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gcgccgatct tggtgtaggt gtcgttggtg g 31
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tctactacaa gaacgtcatc gtcacgaccg ttggtccggg agcacgta 48
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<211> 333
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gctcttcaag gagaacatcg ccccgcacaa gttcaaggcc cacatctact acaagaacgt 60
catcgtcacg accgtgtggt ccgggagcac gtacgcggcc atcacgaacc gcttcacgga 120
ccgcgtgccc gtccccgtgc aggagatcac ggacgtgatc gaccgccgcg gcaagtgcgt 180
ctccaaggcc gagtacgtgc gcaacaacca caaggtgacc gccttcgacc gcgacgagaa 240
ccccgtcgag gtggacctgc gcccctcgcg cctgaacgcg ctcggcaccc gcggctggca 300
caccaccaac gacacctaca ccaagatcgg cgc 333
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accccgagga cgagttcagc agcgacgagg ac 32
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tcaacaggcg gttggcggtc acgccatagt t 31
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ccgaggaggc gccccgctcc ggcttcgacg ttggttccgc gatccgga 48
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accccgagga cgagttcagc agcgacgagg acgacgggct gtacgtgcgc cccgaggagg 60
cgccccgctc cggcttcgac gtctggttcc gcgatccgga gaagccggaa gtgacgaatg 120
gacccaacta tggcgtgacc gccaaccgcc tgttga 156
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atgcccgctg gtggcggtct ttgg 24
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ctacagggcg tcggggtcc 19
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atgcggccct ttctgctgc 19
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ttaagcgggg cgggacatca a 21
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acaagttcaa ggcccacatc tac 23
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gtcygtgaag cggttcgtga t 21
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acgtcatcgt cacgacc 17
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cttccactcg cagctcttct c 21
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gtraagttct cgcgcgagt 19
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ttcgacctga tgccgc 16
Claims (10)
1. one group is used for the nucleic acid of real-time RPA quick detections PRV viruses, it is characterised in that including for detecting PRV's
Sense primer 1, anti-sense primer 1 and exo probes 1；Wherein, the sequence of the sense primer 1 is described as shown in SEQ ID No.1
The sequence of anti-sense primer 1 as shown in SEQ ID No.2, the sequence of the exo probes 1 as shown in SEQ ID No.3, wherein, institute
State the 31st T kilobase marker fluorophor of exo probes 1, the 32nd T kilobase marker fluorescent quenching group, the 31st T alkali
Between base and the 32nd T base be provided with tetrahydrofuran molecule, the exo probes 13 ' end be marked with C3, phosphate group,
Biotin-TEG or amino.
2. nucleic acid as claimed in claim 1, it is characterised in that this group of nucleic acid also positive amplification including PRV Viral diagnosis is produced
Thing sequence 1, the positive amplification Product Sequence 1 includes the sequence as shown in SEQ ID No.4.
3. one group is used for the nucleic acid that real-time RPA quick discriminatings detect the wild poison of PRV and vaccine virus, it is characterised in that including
Nucleic acid as claimed in claim 1 or 2 and sense primer 2, anti-sense primer 2 and exo probes 2 for detecting the wild poison of PRV, it is described
The sequence of sense primer 2 is as shown in SEQ ID No.5, and the sequence of the anti-sense primer 2 is as shown in SEQ ID No.6, the exo
The sequence of probe 2 is as shown in SEQ ID No.7, and the 31st T kilobase marker of the exo probes 2 has different from the exo probes
1 fluorophor, the 32nd T kilobase marker fluorescent quenching group, between the 31st T base and the 32nd T base
Provided with tetrahydrofuran molecule, 3 ' ends of the exo probes 2 are marked with C3, phosphate group, biotin-TEG or amino.
4. nucleic acid as claimed in claim 3, it is characterised in that this group of nucleic acid also includes the positive amplification production of the wild poison detections of PRV
Thing sequence 2, the positive amplification Product Sequence 2 includes the sequence as shown in SEQ ID No.8.
5. the nucleic acid as described in claim 1-4 is any, it is characterised in that the fluorophor is FAM, ROX or TAMARA, institute
Fluorescent quenching group is stated for BHQ1 or BHQ2.
6. the detection kit containing the nucleic acid as described in claim 1-5 is any.
7. detection kit as claimed in claim 6, it is characterised in that the detection kit also includes：RecA recombinases,
Bsu archaeal dna polymerases, single strand binding protein and exo exonucleases, reaction Buffer, magnesium acetate solution.
8. a kind of method that the wild poison of PRV and vaccine virus are detected for real-time RPA quick discriminatings, it is characterised in that specific
Comprise the following steps：
(1) DNA of detection sample is prepared；
(2) DNA described to step (1) carries out isothermal duplication；Wherein, in reaction system, using as described in claim 3
Nucleic acid, is placed in 39 DEG C of isothermal reactions；
(3) 20min is carried out in the isothermal reaction, while collecting fluorescence signal；
(4) result judgement：
If the fluorophor that exo probes described in testing sample 1 is marked has obvious amplification curve, while the exo probes 2 are marked
If fluorophor have obvious amplification curve, that is, be judged as that the wild poison of PRV is positive；
If the fluorophor that exo probes described in testing sample 1 is marked has obvious amplification curve, while the exo probes 2 are marked
Fluorophor without obvious amplification curve, be judged as that PRV vaccine virus is positive；
If the fluorophor that exo probes described in testing sample 1 is marked is without amplification curve, it is judged as that PRV viruses are negative.
9. method as claimed in claim 8, it is characterised in that in the reaction system in the step (2), draw containing the upstream
The final concentration of 200nmol/L of thing 1, anti-sense primer 1, the final concentration of 60nmol/L of the exo probes 1, the sense primer
2nd, the final concentration of 600nmol/L of anti-sense primer 2, the final concentration of 160nmol/L of the exo probes 2.
10. method as claimed in claim 8 or 9, it is characterised in that in the step (2), while setting nuclease-free water is
Negative control；Positive control is used as using comprising the sequence as shown in SEQ ID No.4 and SEQ ID No.8.
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| CN201710556795.8A CN107236825B (en) | 2017-07-10 | 2017-07-10 | Nucleic acid and method for rapidly detecting and distinguishing PRV (porcine reproductive and respiratory syndrome) wild virus and vaccine virus by real-time RPA (RPA) |
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| CN201710556795.8A CN107236825B (en) | 2017-07-10 | 2017-07-10 | Nucleic acid and method for rapidly detecting and distinguishing PRV (porcine reproductive and respiratory syndrome) wild virus and vaccine virus by real-time RPA (RPA) |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107937606A (en) * | 2017-11-23 | 2018-04-20 | 深圳市福田区动物防疫监督所 | A kind of reagent and method for identifying hydrophobia strain and wild type strains |
| CN108179177A (en) * | 2017-12-29 | 2018-06-19 | 博迪泰（厦门）生物科技有限公司 | A kind of kit and detection method of quick detection nucleic acid mutation |
| CN108998505A (en) * | 2018-07-30 | 2018-12-14 | 苏州先达基因科技有限公司 | A kind of gene polymorphism sites detection technique and its kit |
| CN109022614A (en) * | 2018-04-24 | 2018-12-18 | 上海申亚动物保健品阜阳有限公司 | A kind of Taqman real-time fluorescent PCR reagent case and its detection method detecting Pseudorabies virus |
| CN109355428A (en) * | 2018-10-30 | 2019-02-19 | 宁波匠神生物科技有限公司 | Room temperature constant temperature quickly detects primer, probe, reagent and the kit of pseudorabies virus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106048094A (en) * | 2016-07-19 | 2016-10-26 | 金宇保灵生物药品有限公司 | Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe |
| CN106636469A (en) * | 2017-01-12 | 2017-05-10 | 中国人民解放军军事医学科学院微生物流行病研究所 | RPA technology-based marburg virus detection kit and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106048094A (en) * | 2016-07-19 | 2016-10-26 | 金宇保灵生物药品有限公司 | Wild porcine pseudorabies strain and gene-deleted strain dual real-time fluorescence quantification PCR detection kit, primers and probe |
| CN106636469A (en) * | 2017-01-12 | 2017-05-10 | 中国人民解放军军事医学科学院微生物流行病研究所 | RPA technology-based marburg virus detection kit and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937606A (en) * | 2017-11-23 | 2018-04-20 | 深圳市福田区动物防疫监督所 | A kind of reagent and method for identifying hydrophobia strain and wild type strains |
| CN107937606B (en) * | 2017-11-23 | 2020-04-17 | 深圳市福田区动物防疫监督所 | Reagent and method for identifying rabies virus vaccine strain and wild strain |
| CN108179177A (en) * | 2017-12-29 | 2018-06-19 | 博迪泰（厦门）生物科技有限公司 | A kind of kit and detection method of quick detection nucleic acid mutation |
| CN109022614A (en) * | 2018-04-24 | 2018-12-18 | 上海申亚动物保健品阜阳有限公司 | A kind of Taqman real-time fluorescent PCR reagent case and its detection method detecting Pseudorabies virus |
| CN108998505A (en) * | 2018-07-30 | 2018-12-14 | 苏州先达基因科技有限公司 | A kind of gene polymorphism sites detection technique and its kit |
| CN108998505B (en) * | 2018-07-30 | 2022-03-15 | 苏州先达基因科技有限公司 | Gene polymorphism site detection technology and kit thereof |
| CN109355428A (en) * | 2018-10-30 | 2019-02-19 | 宁波匠神生物科技有限公司 | Room temperature constant temperature quickly detects primer, probe, reagent and the kit of pseudorabies virus |
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