CN107254555A - For detecting the primer pair of different subtype canine parvovirus and the combination product of probe - Google Patents
For detecting the primer pair of different subtype canine parvovirus and the combination product of probe Download PDFInfo
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Abstract
The present invention relates to a kind of combination product for being used to detect the primer pair and probe of different subtype canine parvovirus.Any group or whole of the combination product of the primer pair and probe in following primer pair probe combinations：First group：The P of 2 305 P and/or CPV 2a of probe CPV 305；305 R of F, CPV of upstream and downstream primer CPV 305：Shown in 34；Second group：One kind, two kinds or whole in the P of 426 P, CPV 2c of probe CPV 426 P, CPV 2b of 2a 426；426 R of F, CPV of upstream and downstream primer CPV 426.The present invention can be used not only for the types of detection canine parvovirus CPV 2,2a types, 2b types and 2c type strains, moreover it is possible to each strain is made a distinction and identified, there is good application prospect.
Description
Technical field
The present invention relates to field of virus detection, it is used to detect different subtype canine parvovirus in particular to a kind of
The combination product of primer pair and probe.
Background technology
Canine parvovirus can cause the transmissible gastroenteritis of the acute lethal of dog and the subacute myocarditis of pup, the disease
Poison is found in 1978, it is considered to be the variation strain of feline panleucopenia virus, it may be possible to which feline panleucopenia virus sample virus is wild by adapting to
Raw carnivore and evolve come.Although without tangible proof, the presence of FPV and CPV middle precursor virus and FPV are not
Dog can be infected and support this hypothesis always.Originally canine parvovirus is referred to as CPV-2 types, because canine parvovirus is from something lost
(Canine minute virus, CnMV are also known as with the dog piconavirus that is separated before out of dog body in coming into and antigenic characteristic
Canine parvovirus type 1, CPV-1) it is different, so Canine parvovirus type 2 are originally named as, letter
Claim CPV-2.Present dog piconavirus, which is renamed as carnivore, to be won the type of card parvovirus 1 and is under the jurisdiction of rich card parvovirus category, and dog
Parvovirus is under the jurisdiction of former parvovirus category, so CPV refers in particular to canine parvovirus now.CPV at least there occurs 6 compared with FPV
Or the change of 7 amino acid sites, these amino acid sites have impact on virus and the knot of host cell TfR (TfR)
Conjunction ability, so that CPV obtains the ability combined with dog TfR.Another important characteristic is entering for CPV compared with FPV
Change speed faster, the replacement rate that CPV mononucleotide units point is annual has reached 10-4, its gene substitution rate soon with some RNA
The gene substitution rate of virus is similar.
Initial immunization program can aid in the enhancing of dog herd immunity after CPV is popular, reduce dog colony dead
Die rate and containment virus further expands propagation.But host immune pressure has perhaps promoted the appearance of CPV neoantigen types.
Occur in that two kinds of new antigenic types within 1979 and 1984, be respectively designated as CPV-2a types and CPV-2b types, new antigenic type can
To be made a distinction with monoclonal antibody and the CPV-2 types that occur before.Antigenic type CPV-2a and CPV-2b new at present is complete
It is complete to instead of the extensive popular infection in the dog body of countries in the world of CPV-2 types, but CPV-2 types vaccine is still widely being used.
CPV-2a and CPV-2b types there occurs the mutation of 5 or 6 amino acid compared with CPV-2 types on VP2 albumen.CPV-2a with
CPV-2b difference is the change of only 2 amino acid, is N426D and I555V respectively.
426 amino acids are located in the three of the capsid protein Staphylococal Protein A epitope folded on fine dash forward, 426 on mouse piconavirus
The effect of amino acids has been found to relevant with antigen immune escape.Original CPV-2a types are different in the 555th amino acids residue
Leucine, at the 555th is that valine is different from CPV-2/2b types.Along with the 555th amino acids from I (isoleucine) to V
The change of (valine), nearest CPV-2a plants only has difference in the 426th amino acids residue of VP2 capsid proteins and CPV-2b/2c
It is different.
The CPV-2a/2b types found for 1987 and the CPV-2a/2b type differences reported before are on VP2 albumen
297 amino acids there occurs the mutation from serine (Ser) to alanine (Ala), and 297 amino acids residues are located at B on VP2 and resisted
Above former epitope, the mutation in the site changes virus and the affinity of host cell receptor, and is named as new CPV-2a/2b
Type.And 440 amino acids residues from T (threonine) to A (alanine) change wide coverage in isolated strain all over the world
In, but the effect of this site mutation is also indefinite to await further research.267 residues form position to 498 residues
GH close rings between G β-pleated sheets bucket and H β-pleated sheets bucket, the close ring is exposed to the outside of viral capsid proteins, so the section
The change of residue have impact on the plasticity of viral capsid proteins.
2000 on Italian isolated VP2 protein 42s 6 aspartic acid to the variation strain of glutamic acid, later quilt
Referred to as CPV-2c types, quickly CPV-2c types traveled to some other European countries and other continents.Compared with CPV-2 types,
CPV-2a, CPV-2b and CPV-2c etc. new antigenic type virus also constantly expands the pathogenic stronger of dog, and host range
Greatly, including cat can be infected and cause the morbidity of cat.Research finds that CPV-2c types are worldwide extensive popular, and
Usually cause the morbidity of adult dogs seriously, the dog crossed even if vaccine immunity can also fall ill.
Current vaccine immunity is the effective means that dog is effective against parvovirus infections, and inactivated vaccine is merely capable of causing short
The immunity of phase and attenuated live vaccines have been widely used in above this sick immunoprophylaxis, and these vaccines are highly effective
, dog can be protected from the infection of parvovirus, the canine parvovirus virus live vaccine in current CPV-2 types and CPV-2b types source is still
It is very safe, animal can be protected from the attack of virus, and also side reaction also seldom occurs for immune dog.Dog once infects
CPV can only symptomatic treatment, take hyper-immune serum or monoclonal antibody to be treated.Vaccine used is mainly CPV-2 types and CPV-
Single seedling or connection seedling in 2b types source, without CPV-2a types and the vaccine of CPV-2c types.Decaro etc. (2014) serum is popular
Disease is learned research and found, the time of CPV-2 types vaccine and CPV-2b type vaccine immunities dog generation neutralizing antibody is different, Qian Zhe
14d could produce effective neutralizing antibody after immune, and the latter only needs 7d just to reach immunoprotection.Have been reported that recently existing
Vaccine can not completely protect CPV-2c types, it is immune after dog infection CPV and there occurs parvovirus and suffer from diarrhoea.Therefore, nothing
The need for being in order in the research of CPV different subtypes, or it is that the cross protection research between vaccine and clinical treatment provide technology
Support, be required for providing a kind of method of efficient differentiation CPV different subtypes.
In view of this, it is special to propose the present invention.
The content of the invention
The present inventor passes through performing creative labour and substantial amounts of experiment, has obtained can be used in detection detection canine parvovirus
CPV-2 types, 2a types, the primer pair and probe of 2b types and 2c type strains, the primer pair and probe can be used for distinguishing detection dog thin
Small virus CPV-2 types, 2a types, 2b types and 2c type strains, and high specificity, sensitivity height.Thus provide following inventions.
One aspect of the present invention is related to the combination product of a kind of primer pair and probe, and it is selected from following primer pair probe groups
Any group in conjunction or whole：
First group：
Probe CPV-2-305-P and/or CPV-2a-305-P, its nucleotide sequence is respectively such as SEQ ID NO：Shown in 1-2；
Upstream and downstream primer CPV-305-F, CPV-305-R, its nucleotide sequence is respectively such as SEQ ID NO：Shown in 3-4；
Second group：
One kind, two kinds or whole, its nucleosides in probe CPV-2a-426-P, CPV-2b-426-P, CPV-2c-426-P
Acid sequence is respectively such as SEQ ID NO：Shown in 5-7；
Upstream and downstream primer CPV-426-F, CPV-426-R, its nucleotide sequence is respectively such as SEQ ID NO：Shown in 8-9.
The combination of wherein second group primer pair and probe can be used for distinguishing CPV-2/2a types, 2b types, 2c types, but be not used to
Distinguish CPV-2 types, 2a types；The combination of first group of primer pair and probe can be used for distinguishing CPV-2 types and 2a/2b/2c types, but can not
For distinguishing CPV-2a types, 2b types and 2c types.By two groups use cooperatively can be used for detect, quantitative analysis canine parvovirus CPV-2
Any of type, 2a types, 2b types and 2c type strains, or distinguish various strains.
The primer and probe can be synthesized according to the state of the art, and the company system of specialty can also be entrusted standby.
Specifically, the probe carries detectable mark.
It is preferred that, the combination product of primer pair and probe according to any one of the present invention, the probe is itself
Quenching probes.
It is preferred that, the combination product of primer pair and probe according to any one of the present invention, the self-quenched primer
5 ' end mark fluorescents transmitting groups, 3 ' end mark quenching groups.
It is preferred that, the combination product of primer pair and probe according to any one of the present invention, the fluorescent emission group
Selected from FAM, HEX, TAMRA, VIC and JOE.
Probe CPV-2-305-P, CPV-2a-305-P, CPV-2a-426-P, CPV-2b-426-P, CPV-2c-426-P can
To select identical or different fluorescent emission group.
It is preferred that, the combination product of primer pair and probe according to any one of the present invention, the quencher is selected from
TAMRA, BHQ and MGB.
It is furthermore preferred that the end of self-quenched primer 3 ' is combined with minor groove binders (MGB).
Probe CPV-2-305-P, CPV-2a-305-P, CPV-2a-426-P, CPV-2b-426-P, CPV-2c-426-P can
To select identical or different quencher.
Another aspect of the present invention further relates to a kind of composition, and it contains the combination production of primer pair as described above and probe
Product.
The composition can be used in detecting any in canine parvovirus CPV-2 types, 2a types, 2b types and 2c type strains
Kind, or distinguish various strains.
Another aspect of the present invention further relates to a kind of kit, and includes claim primer pair as described above and probe
Combination product, it is preferred that also including the one or more in RT-PCR reaction buffers, reverse transcriptase and Taq enzyme.
It is preferred that, the reverse transcriptase is that efficient reverse transcriptase (can produce big only in 5 minutes from height copy gene
The cDNA of amount, common reverse transcriptase needs half an hour), the Taq enzyme is hot start Taq polymerase.
It is preferred that, the kit according to any one of the present invention, it also includes RNA extracts reagents.
It is preferred that, the kit according to any one of the present invention, it also includes positive control；
One or more in standard strain of the positive control selected from CPV-2, CPV-2a, CPV-2b, CPV-2c；More
It is preferred that, the standard strain is inactivation strain；
Or；
The one kind or many of the positive control in the standard strain containing CPV-2, CPV-2a, CPV-2b, CPV-2c
Plant the plasmid of VP2 sequences.
The kit that the present invention is provided can be used in detection canine parvovirus CPV-2 types, 2a types, 2b types and 2c type strains
It is any, or distinguish various strains.
According to an aspect of the present invention, the invention further relates to the combination product of primer pair as described above and probe, as above
Described composition or kit as described above are being prepared for detecting or differentiating canine parvovirus CPV-2 types, 2a types, 2b types
With the application in the reagent of 2c type strains.
Compared with prior art, beneficial effects of the present invention are：
Fast and convenient, high specificity, sensitiveness height, good reliability, testing cost is low, the time is short, detection efficiency is high, accurate
Property high, false positive it is low.Kit of the present invention can not only detect canine parvovirus CPV-2 types, 2a types, 2b types and 2c type strains,
Each strain can also be made a distinction and be identified, guidance can be provided for the clinical treatment of canine parvovirus, be monitoring, preventing for epidemic situation
Control provides strong technical support, there is good application prospect.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is standard curve when CPV-2 and CPV-2a is identified with first group of primer pair probe combinations；
Fig. 2 is standard curve when CPV-2a, CPV-2b and CPV-2c are identified with second group of primer pair probe combinations；
Fig. 3 is solubility curve when CPV-2 is individually identified with first group of primer pair probe combinations；
Fig. 4 is solubility curve when CPV-2a/2b/2c is identified with first group of primer pair probe combinations；
Fig. 5 is solubility curve when CPV-2, CPV-2a/2b/2c are identified with first group of primer pair probe combinations；
Fig. 6 is solubility curve when CPV-2/2a is identified with second group of primer pair probe combinations；
Fig. 7 is solubility curve when CPV-2b is individually identified with second group of primer pair probe combinations；
Fig. 8 is solubility curve when CPV-2c is individually identified with second group of primer pair probe combinations；
Fig. 9 is solubility curve when CPV-2/2a, CPV-2b and CPV-2c are identified with second group of primer pair probe combinations；
Figure 10 is that multiple TaqMan MGB fluorescent quantitations are carried out to CPV-2, CPV-2a with first group of primer pair probe combinations
The CPV-2 of various concentrations testing result during PCR；
Figure 11 is that multiple TaqMan MGB fluorescent quantitations are carried out to CPV-2, CPV-2a with first group of primer pair probe combinations
The CPV-2a of various concentrations testing result during PCR；
Figure 12 is that multiple TaqMan MGB are carried out to CPV-2a, CPV-2b and CPV-2c with second group of primer pair probe combinations
The CPV-2a of various concentrations testing result during quantitative fluorescent PCR；
Figure 13 is that multiple TaqMan MGB are carried out to CPV-2a, CPV-2b and CPV-2c with second group of primer pair probe combinations
The CPV-2b of various concentrations testing result during quantitative fluorescent PCR；
Figure 14 is that multiple TaqMan MGB are carried out to CPV-2a, CPV-2b and CPV-2c with second group of primer pair probe combinations
The CPV-2c of various concentrations testing result during quantitative fluorescent PCR；
CPV-2/2a, CPV-2a/2b/2c are represented and cannot be distinguished by figure, it is necessary to by passing through two in wherein above-mentioned accompanying drawing
Group could be distinguished with reference to comparison.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
1. sequence analysis
All canine parvovirus VP2 protein sequences are downloaded on GenBank, it is extraordinary dynamic with Gao Yun
Four plants of different subtype strains that thing cause of disease is preserved with immune team：CPV LN15-32 (CPV-2), CPV JL14-1 (CPV-2a),
CPV BJ14-1 (CPV-2b), CPV BJ15-20 (CPV-2c) sequence carries out sequence alignment analysis, VP2 weights using MEGA7
Amino acid and nucleotide site is wanted to be mutated the such as (Reference strains of table 1：CPV-2b, accession number：M38245).
The CPV different genotype VP2 aminopeptidase genes of table 1 acid residue
aAmino acid residue and nucleotide site are using CPV-b strains as with reference to (sequence number：M38245)
bNucleotide site
2. primed probe is designed
MEGA7 compares analysis canine parvovirus VP2 protein sequence, for the mononucleotide difference of the amino acids residues of VP2 305,
CPV-2 vaccine strains and the probe of other wild types are distinguished in design：CPV-2-305-P and CPV-2a-305-P and its upstream and downstream are drawn
Thing：CPV-305-F/CPV-305-R, for the mononucleotide difference of the amino acids residues of VP2 426, CPV-2/2a is distinguished in design
The probe being mutually distinguishable with CPV-2b, CPV-2c：CPV-2a-426-P, CPV-2b-426-P, CPV-2c-426-P, and its up and down
Swim primer：CPV-426-F/CPV-426-R such as tables 2.
The primer of table 2 and probe sequence
3. plasmid construction
3.1 nucleic acid extraction
Tetra- plants of strains of 200ul (CPV LN15-32, CPV JL14-1, CPV BJ14-1, CPV BJ15-20) disease is taken respectively
Toxogen liquid, extracts viral DNA.Operating procedure presses TAKARA MiniBEST Viral RNA/DNA Extraction Kit
Ver.5.0 operating instruction is carried out.
1. 200ul virus stock solution useds are taken, are added in 1.5ml centrifuge tubes.
2. 200ul Buffer VGB, 20ul Proteinase K and 1.0ul Carrier RNA are added, it is fully mixed
It is even in 56 DEG C of water-bath warm bath 10 minutes.
3. Spin Column are placed on Collection Tube, solution is moved in Spin Column, 12000rpm
Centrifugation 2 minutes, abandons filtrate.
4. 500ul Buffer RWA are added into Spin Column, 12000rpm is centrifuged 1 minute, abandons filtrate.
5. 700ul Buffer RWB are added into Spin Column, 12000rpm is centrifuged 1 minute, abandons filtrate.Note：
The absolute ethyl alcohol of designated volume is had been added in Buffer RWB.
6. repeat step is 5..
7. Spin Column are placed on Collection Tube, 12000rpm is centrifuged 2 minutes, abandons filtrate.
8. Spin Column are placed on new 1.5ml Rnase free collection tube, in Spin
Column films centre adds 30-50ul Rnase free dH2O, is stored at room temperature 5 minutes.
9. 12000rpm is centrifuged 2 minutes, eluted dna/RNA.
3.2 PCR are expanded
The DNA extracted using kit enters performing PCR amplification by primer of CPV-up and CPV-down as template.Reference
Primer TaqTM (Ex TaqTM Version 2.0plus dye) (Takara, RR902A) illustrate, prepared by consisting of
PCR reaction solutions
3.3 PCR are purified
1% gel is prepared with the buffer solution (1 × TAE) diluted.5ul PCR primers are taken to add in gel pore, electrophoresis is seen
Examine result.Remaining PCR primer referencePCR Purfication Kit operation instructions are purified.
3.4 connection
The purpose fragment of purifying is connected on PEASY-T1 carriers by following reaction system table 4, reaction condition is 37 DEG C
15min。
The coupled reaction system (5ul) of table 4
The conversion of 3.2 connection products
By connection product, all add first by connection product, all add in the α competent cells of Trans 5, gently clap
Beating tube wall mixes it, after ice bath 30min, is put into 42 DEG C of heat shock 30s, takes out be placed in ice bath 2min immediately；Then add
500ul non-resistant LB nutrient solutions, are positioned over 37 DEG C of 200rpm in shaking table, shake 1h.4000rpm centrifuges 45s, stays 100ul supernatants light
Featheriness plays precipitation, and by bacterium solution even spread and the LB culture plates of ammonia benzyl resistance, culture is inverted in 37 DEG C of constant incubators
Overnight, growing state is observed.Last picking single bacterium colony is put into the LB nutrient solutions containing 100U/ml Amp, is placed in shaking table
37 DEG C of 200rpm about 12h, amplification culture.
3.2 plasmid extractions are identified
Make according to Beijing Quan Shijin Co., Ltds plasmid extraction kit (EasyPure Plasmid MiniPrep Kit)
Plasmid extraction is carried out with specification, performing PCR checking, and Song Kumei biotech firms sequencing analysis are entered using CPV-up/CPV-down.
This research builds four kinds of plasmids altogether, is respectively designated as CPV LN15-32, CPV JL14-1, CPV BJ14-1, CPV BJ15-20
4. standard curve is set up
By CPV LN15-32, CPV JL14-1, CPV BJ14-1, tetra- kinds of standard plasmid distilled waters 10 of CPV BJ15-20
Be serially diluted into 8 gradients again, make its final concentration of 1 × 109Copies/ul~1 × 102copies/ul.As template, in
On ABI QuantStudioTM Design fluorescent quantitation quantitative instruments, expanded according to the system that optimal prominent light is quantitative,
Standard curve is automatically generated in software kit after the completion of amplification.As a result respectively as depicted in figs. 1 and 2.
Carried out respectively with first group and second group of primer individually and such as Fig. 3-figure respectively of solubility curve result during multiplex PCR
Shown in 9.
5. specific test
With CPV LN15-32, CPV JL14-1, CPV BJ14-1, the standard plasmid of tetra- plants of strains of CPV BJ15-20 and
The viral hepatitis infectiosa canis virus (CAV) of DNA and other dog sources, canine coronavirus (CCV), the DNA or cDNA of CDV (CDV) are
Template, fluorescent quantitation is carried out under optimization reaction condition, identifies its specificity.
305,426 probes and primer can carry out detection amplification to the CPV plasmids and DNA of above-mentioned 4 kinds of genotype, and right
CAV, CCV, CDV DNA or cDNA are without amplification, and showing the primer and probe of this experiment has good specificity.
6. sensitivity tests
By CPV LN15-32, CPV JL14-1, CPV BJ14-1, tetra- kinds of standard plasmid distilled waters 10 of CPV BJ15-20
Be serially diluted into 7 gradients again, make its final concentration of 1 × 106Copies/ul~1 × 100copies/ul.As template, in
On ABI QuantStudioTM Design fluorescent quantitation quantitative instruments, substance TaqMan MGB quantitative fluorescent PCRs are carried out respectively, really
Regular inspection goes out lower limit；By CPV LN15-32 and CPV JL14-1 standard plasmids 1:1 mixing, 10 times of dilutions of distilled water, makes its final concentration
For 5 × 106Copies/ul~5 × 100Copies/ul, carries out multiple TaqMan MGB glimmering with first group of primer pair probe combinations
Fluorescent Quantitative PCR, it is determined that detection lower limit, as a result respectively as shown in Figure 10 and Figure 11；By CPV JL14-1 and CPV BJ14-1 and CPV
BJ15-20 standard plasmids 1：1：1 mixing, the dilution of 10 times of distilled water, make its final concentration of 3.3 × 106Copies/ul~3.3 ×
100Copies/ul, multiple TaqMan MGB quantitative fluorescent PCRs are carried out with second group of primer pair probe combinations, it is determined that detection lower limit
As a result respectively as shown in figs. 12-14.
Figure 10 CPV-2；1×101copies/ul
Figure 11 CPV-2a；1×102copies/ul
Figure 12 CPV-2a；3.3×101copies/ul
Figure 13 CPV-2b；3.3×101copies/ul
Figure 14 CPV-2c；3.3×102copies/ul
7. replica test
By CPV LN15-32, CPV JL14-1, CPV BJ14-1, tetra- kinds of recombinant plasmid standard items of CPV BJ15-20,10
After times doubling dilution, high, medium and low three concentration gradients (10 are taken8copies/ul、106copies/ul、104Copies/ul), it is first
First single plasmid standard is carried out respectively in substance fluorescent quantitation group PCR groups and replica test between group, then by plasmid control
Product mixing carries out differentiating respectively vaccine strain and street strain, and differentiates in CPV-2a/2b/2c multiple fluorescence quantitative group and weight between group
Renaturation test, according to value calculating group in and between-group variation coefficient, identify its stability.
The above-mentioned experiment coefficient of variation is between 0.2%-2.5%, with good repeatability.
8. clinical practice is tested
Using the TaqMan MGB multiple fluorescence quantitative PCRs set up, the collaurum of the regional censorship such as Hebei, Beijing is tried
Paper slip detection canine parvovirus positive fecal specimens detected, and binding sequence measure with evaluate this method practicality and
Coincidence rate.
Clinical 114 parts of CPV pathological material of diseases are carried out fluorescence quantitative PCR detection and carry out sequencing to meet experimental result such as
Under.Fluorescent quantitative PCR result and sequencing result coincidence rate are 100%, in 114 parts of positive pathological material of diseases of detection, CPV-2,2a, 2b
Be respectively 2 with 2c types, 57,25 and 30 parts.
The PCR system of table 3 (50ul)
Amplification response procedures are in advance：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 1min, 55 DEG C of annealing 1min, 72 DEG C of extensions
2min；Circulation 35 times；72 DEG C extend 10min eventually.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, but it will be understood by those within the art that：Its
The technical scheme described in foregoing embodiments can still be modified, or to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>Gao Yun
<120>For detecting the primer pair of different subtype canine parvovirus and the combination product of probe
<160> 9
<170> PatentIn version 3.3
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actcctatat caccaaagtt ag 22
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actcctatat aaccaaagtt agta 24
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cgttgcctca atctgaagga gcta 24
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ttgcccattt gagttacacc acgt 24
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cctgtaacaa atgataatgt attgc 25
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cttcctgtaa cagatgataa tgtatt 26
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cttcctgtaa cagaagataa tgtatt 26
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aggaagatat ccagaaggag attgga 26
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ccaattggat ctgttggtag caataca 27
Claims (10)
1. the combination product of a kind of primer pair and probe, its any group or whole in following primer pair probe combinations：
First group：
Probe CPV-2-305-P and/or CPV-2a-305-P, its nucleotide sequence is respectively such as SEQ ID NO：Shown in 1-2；
Upstream and downstream primer CPV-305-F, CPV-305-R, its nucleotide sequence is respectively such as SEQ ID NO：Shown in 3-4；
Second group：
One kind, two kinds or whole, its nucleotides sequence in probe CPV-2a-426-P, CPV-2b-426-P, CPV-2c-426-P
Row are respectively such as SEQ ID NO：Shown in 5-7；
Upstream and downstream primer CPV-426-F, CPV-426-R, its nucleotide sequence is respectively such as SEQ ID NO：Shown in 8-9.
2. the combination product of primer pair according to claim 1 and probe, it is characterised in that the probe is that itself quenches
Go out probe.
3. the combination product of primer pair according to claim 2 and probe, it is characterised in that the self-quenched primer
5 ' end mark fluorescent transmitting groups, 3 ' end mark quenching groups.
4. the combination product of primer pair according to claim 3 and probe, it is characterised in that the fluorescent emission group choosing
From FAM, HEX, TAMRA, VIC and JOE.
5. the combination product of primer pair according to claim 3 and probe, it is characterised in that the quencher is selected from
TAMRA, BHQ and MGB.
6. a kind of composition, it contains the combination product of primer pair described in claim any one of 1-5 and probe.
7. a kind of kit, and comprising the primer pair and the combination product of probe described in claim any one of 1-5, preferably also wrap
Include the one or more in RT-PCR reaction buffers, reverse transcriptase and Taq enzyme.
8. kit according to claim 7, it is characterised in that it also includes RNA extracts reagents.
9. kit according to claim 7, it is characterised in that it also includes positive control；
One or more in standard strain of the positive control selected from CPV-2, CPV-2a, CPV-2b, CPV-2c；It is preferred that
, the standard strain is inactivation strain；
Or；
One or more VP2s of the positive control in the standard strain containing CPV-2, CPV-2a, CPV-2b, CPV-2c
The plasmid of sequence.
10. composition described in the combination product of primer pair and probe described in claim any one of 1-5, claim 6 or
Kit described in claim any one of 7-9 is being prepared for detecting or differentiating canine parvovirus CPV-2 types, 2a types, 2b types
With the application in the reagent of 2c type strains.
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