CN105803112A - Primer, probe and kit for detecting canine parvovirus and detection method - Google Patents
Primer, probe and kit for detecting canine parvovirus and detection method Download PDFInfo
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Abstract
The invention relates to a primer, probe and kit for detecting canine parvovirus and a detection method.According to the fluorogenic quantitative PCR primer for detecting canine parvovirus, an upstream detection primer CPV-F has the nucleotide sequence as shown in the SEQ ID NO.1, and a downstream detection primer CPV-R has the nucleotide sequence as shown in the SEQ ID NO.2.The probe has the nucleotide sequence as shown in the SEQ ID NO.3.The kit comprises the primer and the probe.The fluorogenic quantitative PCR primer, probe and kit for detecting canine parvovirus have the advantages that operation is easy, cost is low, sensitivity is high and specificity is high, and can guarantee clinical detection.
Description
Technical field
The invention belongs to the detection field of virus, particularly relate to a kind of one-step method and directly detect the fluorescence of Canine Parvovirus calmly
The amount primer of PCR, probe, test kit and detection method thereof.
Background technology
Canine Parvovirus (Canine Parvovirus, CPV) is to cause Canis animals with violent vomiting, hemorrhagic intestinal
Inflammation, apyetous myocarditis and leukocyte are significantly reduced to the deadly infectious disease poison of principal character.Canine parvovirus disease is a kind of
There is the deadly infectious disease of high degree in contact, be characterized with vomiting, diarrhoea and leukopenia.Natural infection Canine Parvovirus
Case often occurs, even if also can fall ill after CPV vaccine immunity, the mortality rate of pup is the highest (20%-30%).Along with dog is raised
Supporting being significantly increased of the fast development of industry, especially experimental dog and pet dog breeding amount, it is the tightest that dog infects parvovirus infection
Weight, causes great economic loss to dog breeders industry.Canine Parvovirus is single minus-strand dna virus, without peplos, to physics
Chemical factor has the strongest resistance, and virus can be survived for a long time in feces, and pathogenicity will not reduce, and infectiousness is very
By force.Sick dog is the main source of infection, and animal is mainly fallen ill by contact infection.
Canine Parvovirus is divided into 1 type and 2 types, and wherein 2 types are divided into again tri-hypotypes of 2a, 2b and 2c, CPV-2a, 2b and CPV-
2 compare, the most several amino acid whose differences.Canine Parvovirus 2 type (CPV-2) is to cause the main pathogen of canine parvovirus disease micro-
Biological.Virion diameter about 20-25nm, gene group leader 5323bp, have two main opening code-reading frames (ORF): NS reading code
Frame and S reading frame, the former product NS1 and NS2 two kinds non-knot hook albumen, for gene replication with transcribe required, the latter mainly compiles
Code capsid protein VP1 (70-90kD), VP2 (62-76kD) and VP3 (39-69kD).VP2 is the main one-tenth constituting viral capsid
Point, determine host range and the antigenicity of virus.
The method of detection Canine Parvovirus mainly observes clinical symptoms at present, and method for detecting virus has test strips, also has
Report normal PCR and quantitative fluorescent PCR, but the accuracy of test strips is the most poor, and traditional PCR method is the longest and clever
Sensitivity is on the low side.
Although but the quantitative fluorescent PCR that presently, there are compares the method sensitivity of traditional PCR method and ELISA test strip
Improve with accuracy, but remain the defects such as testing cost is higher, the longest, operation is complicated.Such as, fixed at fluorescence
During amount PCR, need to utilize nucleic acid extraction liquid sample carries out pretreatment and extracts viral nucleic acid (such as: DNA and RNA etc.), as
The really poor quality of nucleic acid extraction, can affect follow-up experiment, and especially RNA is susceptible to degraded, so extract
Step is the most complex and strict.
Also have and utilize bio-barcode to carry out the method detecting Canine Parvovirus, but the method needs, and to be used for detecting dog thin
The bio-barcode of small virus and bar code with the use of complementary probe NP chain and nucleotide sequence with the use of, and
The method operation is more complicated, and relatively costly, is unfavorable for the detection of large sample amount and epidemiological investigation.
Summary of the invention
In view of the limitation of current canine parvovirus virus detection method, the present invention establishes the one-step method of detection Canine Parvovirus
Quantitative PCR detection method, has the advantages such as easy and simple to handle, low cost, highly sensitive and specificity be good, carries for Clinical detection
For ensureing.
The technical scheme is that
The primer detected for Canine Parvovirus, including:
Upstream detection primer CPV-F includes the nucleotide sequence shown in SEQ ID NO.1, shown in described SEQ ID NO.1
Nucleotides sequence be classified as 5 '-GAAGGTATAAATTCACCAGGTTGC-3 '；
Downstream detector primer CPV-R includes the nucleotide sequence shown in SEQ ID NO.2, shown in described SEQ ID NO.2
Nucleotides sequence be classified as: 5 '-GTGCAAGGTCCACTACGTCC-3 '.
Preferably, described upstream detection primer CPV-F is the nucleotide sequence shown in SEQ ID NO.1, and detected downstream is drawn
Thing CPV-R is the nucleotide sequence shown in SEQ ID NO.2.
Utilize above-mentioned primer can extend a length of 112bp of purpose fragment.
The invention has the beneficial effects as follows: when utilizing above-mentioned primer to detect, there is good specificity.Only need to be to sample
Carry out simple pretreatment, be not required to extract nucleic acid, directly detect with sample, have that step is easier, cost is lower, operation by mistake
Less, the most shorter, the sensitivity more advantages of higher of difference, it is possible to realize completing detection quickly and easily.
The present invention also provides for a kind of probe for Canine Parvovirus detection, including the nucleotide shown in SEQ ID NO.3
Sequence, the nucleotides sequence shown in described SEQ ID NO.3 is classified as 5 '-AGACACAAGCGGCAAGCAATCCTC-3 '.
Preferably, described probe is the nucleotide sequence shown in SEQ ID NO.3.
Inventor, by being repeatedly detected, screen and verifying, obtains this probe unexpectedly, has specificity more high excellent
Point.
Further, described probe 5 ' end there is fluorescent reporter group, described fluorescent reporter group selected from FAM, TET,
Any one in VIC, JOE, HEX, Cy3 and Cy5.
Above-mentioned further scheme is used to provide the benefit that: to have suitable, efficient (strong) and excite and transmitted wave
Long scope, and there is the advantages such as good light stability.
Further, 3 ' ends of described probe have quenching group, and described quenching group is selected from BHQ-1, BHQ-2, TAMRA.
Above-mentioned further scheme is used to provide the benefit that: for receiving the fluorescence signal that fluorescent reporter group is launched, to adopt
With the quenching group of mentioned kind, there is sensitivity advantages of higher.
Further, the 5 ' of described probe are held as FAM, and 3 ' hold as BHQ-1.Probe (name nominating is CPV-probe) with
The sequence of fluorescent reporter group and quenching group is: 5 ' FAM-AGACACAAGCGGCAAGCAATCCTC-3 ' BHQ-1.
Above-mentioned further scheme is used to provide the benefit that: BHQ-1 itself does not produce fluorescence, and fluorescence background is low, sensitivity
Improve further.
The present invention also provide for a kind of for Canine Parvovirus detection test kit, this test kit include above-mentioned primer and/
Or above-mentioned probe.
Take technique scheme have the beneficial effect that above-mentioned probe and above-mentioned primer with the use of, when detection, only need
Sample is carried out simple pretreatment, is not required to extract nucleic acid, directly detects with sample, have that step is easier, cost is lower,
Operating error is less, the most shorter, sensitivity more advantages of higher, it is possible to realize completing detection quickly and easily.
Further, one or more during this test kit also includes reactant liquor A mixture, positive control and negative control；
Described reactant liquor A mixture includes Taq enzyme and PCR reaction buffer；
Described positive control is to include the DNA fragmentation of CPV-NS gene, plasmid or bacterial strain；
Described negative control is aquesterilisa.
GenBank Serial No. M19296 of described CPV-NS gene.
DNA fragmentation, plasmid or bacterial strain including CPV-NS gene can use the conventional method of this area to prepare.Such as:
PCR amplification can be carried out, it is thus achieved that DNA fragmentation according to the primers of CPV-NS gene；Can be by the DNA of acquisition with suitable
Carrier connect, it is thus achieved that containing the plasmid of this gene；Can also this plasmid be proceeded in bacterial strain, it is thus achieved that containing the bacterium of this gene
Strain.
The present invention provides a kind of method that Canine Parvovirus detects, and comprises the following steps:
1) gather sample and carry out pretreatment；
2) quantitative fluorescent PCR expands:
By above-mentioned primer, probe, Taq enzyme, PCR reaction buffer and step 1) sample mix, prepare PCR reactant
System, obtains experimental group；
Arranging positive controls and negative control group, the sample in positive controls is positive control, negative control simultaneously
Sample in group is negative control；Afterwards experimental group, positive controls and negative control group are carried out quantitative fluorescent PCR to carry out
Amplification；
3) interpretation of result and judgement:
A) the Ct value that Ct value is blank and positive controls meeting negative control group in same single test is less than 30, says
Effectively, otherwise this experiment is invalid in this experiment bright；
B) in the case of step a) illustrates that this experiment is effective,
If the amplification curve of institute's test sample product is the most S-type or Ct value is blank, then experimental result is judged to feminine gender；
If the amplification curve of institute's test sample product is S-type and Ct value ＜ 36, then experimental result is judged to the positive；
If 40 ＞ Ct value >=36, then for experiment gray area, need to repeating to test once, if repeating experimental result growth curve being
S type and Ct value ＜ 40, it is determined that for the positive, be otherwise judged to feminine gender.
Further, step 1) in, described sample includes solid sample and/or liquid sample, the pre-place of described liquid sample
Reason method takes supernatant after centrifugal, and the preprocess method of described solid sample is for adding centrifuging and taking supernatant after suspension mixes.
Above-mentioned further scheme is used to provide the benefit that:
First clinical samples is centrifuged by detection method of the present invention, takes supernatant, then directly utilizes the spy of the present invention
Pin and said method carry out quantitative PCR detection, only need a step i.e. can determine whether whether there is Canine Parvovirus in testing sample, are one
Footwork directly detects the detection method of Canine Parvovirus.This detection method detection efficiency height, specificity and sensitivity are good, it is possible to full
Foot Clinical detection and the purpose of epidemic situation monitoring.
Further, step 2) in quantitative fluorescent PCR carry out the condition of amplified reaction and be:
Above-mentioned further scheme is used to provide the benefit that: suitably amplification condition advantageously ensures that the accuracy of amplification.
The present invention also provides for above-mentioned primer, probe and test kit for Canine Parvovirus detection at detection Canine Parvovirus
In application.
Accompanying drawing explanation
Fig. 1 is the sensitivity experiments of one-step method fluorescence quantitative PCR detection CPV, is from left to right followed successively by 100、10-1、10-2、
10-3、10-4、10-5、10-6Standard virus liquid amplification；
Fig. 2 is quantitative fluorescent PCR product electrophoretogram, and wherein, M is DNA molecular amount maker；N is negative quality-control product, remaining
It is 100、10-1、10-2、10-3、10-4、10-5、10-6The detection sample of standard virus liquid.
Fig. 3 is that regular-PCR method detects CPV virus results, and wherein M is DNA molecular amount maker；D is positive quality control product；N
For negative quality-control product, remaining is 100、10-1、10-2、10-3、10-4、10-5、10-6The detection sample of standard virus liquid.
Fig. 4 is the specificity experiments of one-step method fluorescence quantitative PCR detection CPV, and as seen from Figure 4, CPV is positive (bag
Include strong positive and the weak positive), CDV, CAV-II and negative quality-control product are feminine gender；
Fig. 5 is the CPV in one-step method fluorescence quantitative PCR detection dog stool sample, and from left to right, second is positive charge
Product, remaining 29 positive is 29 parts of sick dog stool samples, and 5 feminine genders are respectively 4 normal dogs stool samples and 1 negative Quality Control
Product.
Fig. 6 is the CPV in one-step method fluorescence quantitative PCR detection dog serum specimen, and 22 positive serum specimen all detect
Canine Parvovirus, negative control is normal dogs serum specimen, and testing result is negative.
Detailed description of the invention
Being described principle and the feature of the present invention below in conjunction with accompanying drawing, example is served only for explaining the present invention, and
Non-for limiting the scope of the present invention.The person skilled in the art of this area the present invention is made according to present invention some are non-
Improvement and the adjustment of essence still fall within protection scope of the present invention.
Embodiment 1
1, specific primer and the design of probe
According to the nucleotide sequence of the Canine Parvovirus retrieved on Genbank, it is designed for detecting Canine Parvovirus
Primer and probe, its sequence is as follows:
Forward primer (CPV-F): 5 '-GAAGGTATAAATTCACCAGGTTGC-3 ', as shown in SEQ ID NO.1；
Downstream primer (CPV-R): 5 '-GTGCAAGGTCCACTACGTCC-3 ', as shown in SEQ ID NO.2；
Fluorescent probe (CPV-probe): 5 ' FAM-AGACACAAGCGGCAAGCAATCCTC-3 ' BHQ-1.
Above-mentioned primer and fluorescent probe are synthesized by Shanghai JiMa pharmacy Technology Co., Ltd.
The concentration of forward primer, downstream primer and fluorescent probe is respectively 0.2 μM, 0.2 μM and 0.2 μM.
Utilize a length of 112bp of the purpose fragment of above-mentioned primer and probe amplification.
2, test kit constituent
The test kit of the present invention includes that constituent includes four parts: reactant liquor A mixture (Mix-A), reactant liquor B mix
Thing (Mix-B), positive quality control product and negative quality-control product.
Wherein, Mix-A is Taq enzyme, PCR reaction buffer and the mixture of aquesterilisa；Mix-B includes primer CPV-F, draws
Thing CPV-R, fluorescent probe CPV-probe and aquesterilisa.
Concentration and the volume of each component of preparation are as follows:
Wherein, Mix-A is Taq enzyme (concentration is 5U/ μ L, and volume is 0.5 μ L), PCR reaction buffer (volume is 25 μ L)
Mixture with aquesterilisa (volume is 2.5 μ L)；
Mix-B includes primer CPV-F (concentration is 10 μMs, and volume is 0.5 μ L), (concentration is 10 μMs to primer CPV-R, volume
Be 0.5 μ L), fluorescent probe CPV-probe (concentration is 10 μMs, and volume is 0.5 μ L) and aquesterilisa (15.5 μ L).
Taq enzyme and PCR reaction buffer are purchased from TIANGEN Biotech (Beijing) Co., Ltd., and aquesterilisa is by Millipore
The preparation of ultra-pure water instrument high temperature and high pressure steam sterilizing, positive quality control product (i.e. positive control) is the plasmid containing CPV-NS gene,
Negative quality-control product (i.e. negative control) is aquesterilisa.
The construction method reference of the plasmid containing CPV-NS gene: Canine Parvovirus NS1 non-structural protein can inducing cell
Apoptosis, it is intelligent that peak Zhang Kaochen opened by Pan Hong beautiful Zhong Fei Pan Su quick Li Xiu brocade, microorganism journal, the 3rd phase: 367-373 in 2012.
3, the collection of specimen and pretreatment
The method of the invention is suitable for specimen types and includes the tissue of dog, serum, Secretory product (such as feces etc.).
The compound method of PBS: 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na2HPO4)
With 0.24g potassium dihydrogen phosphate (KH2PO4), adjust pH 7.2, be settled to 1L.
The collection of tissue specimen: aseptically take and organize about about 100-200mg, is placed in 1.5mL cleaning EP pipe,
Adding equimultiple volume PBS washing tissue, room temperature vortex concussion 1min, 10000rpm are centrifuged 2min, take supernatant and preserve to be checked；
The collection of serum: with disposable sterilized injector extraction by inspection animals iv blood 3-5mL, leaving and taking serum, preservation is treated
Inspection.
The collection of Secretory product: gather under aseptic condition, if liquid sample direct centrifuging and taking supernatant；If solid mark
The PBS of this addition equimultiple volume, washing, vortex 1min, 10000rpm are centrifuged 2min, take supernatant, preserve to be checked.
The specimen of above-mentioned collection should censorship as early as possible, or be stored in-20 DEG C.
4, fluorescent quantitative PCR
Reaction system is formulated as follows: Mix-A 28 μ L, Mix-B 17 μ L, sample 5uL.
PCR reaction condition is: hold 95 DEG C of 5s of 95 DEG C of 10s, cycle, 60 DEG C of 15s, 40 circulations.
Experimental group: sample is the sample to be tested that step 3 obtains；
Positive controls: sample is positive quality control product, i.e. contains the plasmid of CPV-NS gene.
Negative control group: sample is negative quality-control product, i.e. water.
5, interpretation of result and judgement
After reaction terminates, it is analyzed according to the software of each quasi-instrument, regulates threshold value, make negative control Ct value not go out as far as possible
What numerical value incumbent.
Experiment all should arrange positive control and negative control every time.
First, in same single test, meet following two condition, i.e. condition 1 and condition 2 simultaneously:
Condition 1: negative control group is negative and Ct value is blank；
Condition 2: positive controls is positive and Ct value ＜ 30.
This experiment is described effectively on the premise of meeting condition 1 and condition 2 at the same time, otherwise, illustrates that this experiment is invalid,
Experiment need to be re-started.
Secondly, on the premise of experiment effectively, continue to judge have three kinds of situations:
(1) negative findings judges: the amplification curve of institute's test sample product is the most S-type or Ct value is as blank, then experimental result judges
For feminine gender.
(2) positive findings judges: the amplification curve of institute's test sample product is S-type, and Ct value ＜ 36, then experimental result is judged to sun
Property.
(3) experiment gray area: if 40 ＞ Ct value >=36, then for experiment gray area, need to repeat to test once, if repeating real
Testing result growth curve is S type and Ct value ＜ 40, it is determined that for the positive, be otherwise judged to feminine gender.
Embodiment 2 sensitivity experiment
Take Chengdu military area command disease prevention and control center preserve Canine Parvovirus separation strain (this separation strain can be for public institute
Obtain), the reference information reference literature of this Canine Parvovirus separation strain: the isolation identification of Canine Parvovirus, Du Qiang Qiu Wei model spring
The raw Zheng Ying Zhang Fuqiang of water Liu Hua Lizuo, volume 30 the 3rd phase 55-58 page in 2009.Virus titer titration TCID50 is 10-4, successively
10 times of doubling dilutions, extension rate be respectively as follows: dilution 1 times, 10 times, 100 times, 1000 times, 10000 times, 100000 times,
1000000 times, the solution after dilution is designated as 10 respectively0、10-1、10-2、10-3、10-4、10-5、10-6, carry out sensitivity experiment,
Specific experiment step is with reference to above-described embodiment 1.
One-step method CPV fluoroscopic examination sensitivity experiment result is shown in such as Fig. 1, and amplification curve is followed successively by 10 from left to right0、10-1、
10-2、10-3、10-4、10-5、10-6Testing result.Amplified production detects with agarose gel electrophoresis further, and result is shown in Fig. 2,
But testing result difference is inconspicuous, it is seen that for the virus of trace, PCR primer agarose gel electrophoresis detects, its result
It is difficult to judge.And after quantitative fluorescent PCR arranges threshold value, accurately can be judged by Ct value, it is better than regular-PCR method.
The most same viral specimen, uses the most more common VP2 primer regular-PCR method to examine further
Survey.
The forward primer of VP2: 5 '-AACGGATGGGTGGAAATCAC-3 ', sequence is as shown in SEQ ID NO.4；
The downstream primer of VP2: 5 '-TAATAGTAGCTTCAGTAATA-3 ', sequence is as shown in SEQ ID NO.5.
The target product size utilizing the forward primer of VP2 and the downstream primer amplification of VP2 is 826bp, and PCR primer is through fine jade
Sepharose electrophoresis.Testing result is shown in that Fig. 3, result show, regular-PCR method detection sensitivity is more fixed than the fluorescence that the present invention sets up
Amount PCR method is low 100 times.
Result confirms that this method detection sensitivity is higher, is 10 for TCI D50-4Virus, dilute 1,000,000 times remain to inspection
Measure, be fully able to meet the detection of clinical dog specimen, and its sensitivity is substantially better than regular-PCR method.
Inventor also uses commercially available Dutch Intervet canine distemper-tiny bigeminy vaccine to carry out sensitivity experiment, also can obtain
To consistent conclusion.Method detection sensitivity of the present invention is higher, is fully able to meet the detection of clinical dog specimen, and
Its sensitivity is substantially better than regular-PCR method.
Embodiment 3 specificity experiments
According to the detection method of above-described embodiment 1, the Canine Parvovirus separated from zoological specimens respectively, canine distemper disease
Poison, hepatitis infectiosa canis virus-II strain are testing sample, and above-mentioned testing sample is maintained in the experiment of Chengdu military area command disease prevention and control center
Room, can be for the public to obtain, and concrete details are referred to document: the clone of Protein Fragment of Canine distemper virus Yunnan Strain H gene
With expression, Nie Fupingli makees the raw the most imperial expensive big Liao Jin of Qiu Wei Zhang Fuqiang Zhang Lianjiang Song Gui and appoints the beautiful jade-like stone remaining side virtue strong model of Zhang Quanpeng Wang Ling
Spring water, animal medicine progress the 1st phase of volume 29 in 2008: 2-12 page.CDV, CPV, CAV tri-the experiment immunization of DNA vaccination grind
Studying carefully, expensive big Lee of the red Nie Fupinglong of Zheng Ying Qiu Wei Li Li makees raw Zhang Fuqiang Li Jiang model spring water, Yunnan University's journal: natural science edition,
2007,29 (5): 529～535.
The negative control simultaneously setting up water to be template, carries out confirmatory experiment.Result confirms that upstream detection of the present invention is drawn
Thing CPV-F, downstream detector primer CPV-R, fluorescent probe and detection method have good specificity, false sun do not occur
Property, accuracy rate is 100%, and experimental result is shown in Fig. 4.As seen from Figure 4, testing result Canine Parvovirus is positive, canine distemper
Virus and hepatitis infectiosa canis virus-II and negative quality-control product are negative.
The detection of CPV in embodiment 4 dog stool sample
Gather feces (5 parts) and the feces (2 parts) of normal dogs of sick dog, add the PBS solution of equimultiple volume, mixing
1min, 10000rpm are centrifugal, take supernatant 5uL, and the method such as reaction system as described in above-described embodiment 1 and reaction condition carries out one
Footwork PCR expands, and result is shown in Fig. 5.As seen from Figure 5,5 parts of sick dog stool samples and positive quality control product testing result are judged to
The positive, 2 parts of normal dogs feces and negative quality-control product testing result are judged to feminine gender.
The feces of the feces and 4 parts of normal dogs that have further carried out 29 parts of sick dogs detects, and accuracy rate is 100%.
The detection of CPV in embodiment 5 dog serum specimen
Gather sick dog and the serum of normal dogs, take supernatant 5uL, the reaction system as described in above-mentioned steps (4) and reaction bar
Part carries out the amplification of one-step method PCR, and result is shown in Fig. 6.As seen from Figure 6,1 part of sick dog serum specimen and positive quality control product are all examined
Surveying result and be judged to the positive, 1 part of normal dogs serum specimen result is judged to feminine gender.
The serum of the serum and 4 parts of normal dogs that have further carried out 22 parts of sick dogs detects, and accuracy rate is 100%.
These results suggest that fluorescent probe of the present invention, primer and method, there is higher sensitivity and good
Specificity, and through verification experimental verification is repeated several times, there is well repeatability and stability.And through clinical dog stool sample
Inspection, Detection results is preferable.
This method has fast and convenient, is not required to extract nucleic acid, directly takes zoological specimens, i.e. can be used for examining through simple process
Survey.Through experimental verification repeatedly, the method has good sensitivity, specificity and repeatability.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
。
Claims (8)
1. for the primer of Canine Parvovirus detection, it is characterised in that including:
Upstream detection primer CPV-F includes the nucleotide sequence shown in SEQ ID NO.1, the core shown in described SEQ ID NO.1
Nucleotide sequence is 5 '-GAAGGTATAAATTCACCAGGTTGC-3 '；
Downstream detector primer CPV-R includes the nucleotide sequence shown in SEQ ID NO.2, the core shown in described SEQ ID NO.2
Nucleotide sequence is: 5 '-GTGCAAGGTCCACTACGTCC-3 '.
2. mate the probe for Canine Parvovirus detection used with primer described in claim 1, it is characterised in that include
Nucleotide sequence shown in SEQ ID NO.3, the nucleotides sequence shown in described SEQ ID NO.3 is classified as 5 '-
AGACACAAGCGGCAAGCAATCCTC-3′。
The most according to claim 2 for the probe of Canine Parvovirus detection, it is characterised in that 5 ' ends of described probe have
Fluorescent reporter group, any one in FAM, TET, VIC, JOE, HEX, Cy3 and Cy5 of described fluorescent reporter group.
4. according to the probe detected for Canine Parvovirus described in Claims 2 or 3, it is characterised in that 3 ' ends of described probe
Having quenching group, described quenching group is selected from BHQ-1, BHQ-2, TAMRA.
5. for the test kit of Canine Parvovirus detection, it is characterised in that this test kit includes the primer described in claim 1
And/or the probe described in any one of claim 2-4.
The most according to claim 5 for the test kit of Canine Parvovirus detection, it is characterised in that this test kit also includes instead
Answer one or more in liquid A mixture, positive control and negative control；
Described reactant liquor A mixture includes Taq enzyme and PCR reaction buffer；
Described positive control is to include the DNA fragmentation of CPV-NS gene, plasmid or bacterial strain, the GenBank Serial No. of this gene
M19296；
Described negative control is aquesterilisa.
7. the method for a Canine Parvovirus detection, it is characterised in that comprise the following steps:
1) gather sample and carry out pretreatment；
2) quantitative fluorescent PCR expands:
By probe, Taq enzyme, PCR reaction buffer and step described in the primer described in claim 1, any one of claim 2-4
Rapid 1) sample mix, prepares PCR reaction system, obtains experimental group；
Arranging positive controls and negative control group, the sample in positive controls is positive control, in negative control group simultaneously
Sample be negative control；Afterwards experimental group, positive controls and negative control group are carried out quantitative fluorescent PCR to expand；
3) interpretation of result and judgement:
A) the Ct value that Ct value is blank and positive controls meeting negative control group in same single test is less than 30, and this is described
Secondary experiment is effective, and otherwise this experiment is invalid；
B) in the case of step a) illustrates that this experiment is effective,
If the amplification curve of institute's test sample product is the most S-type or Ct value is blank, then experimental result is judged to feminine gender；
If the amplification curve of institute's test sample product is S-type and Ct value ＜ 36, then experimental result is judged to the positive；
If 40 ＞ Ct value >=36, then for experiment gray area, need to repeat to test once, if repeating experimental result growth curve is S type
And Ct value ＜ 40, it is determined that for the positive, otherwise it is judged to feminine gender.
The method of a kind of Canine Parvovirus detection, it is characterised in that step 1) in, described sample
Including solid sample and/or liquid sample, the preprocess method of described liquid sample takes supernatant, described solid sample after centrifugal
Preprocess method for add suspension mixing after centrifuging and taking supernatant；Step 2) in quantitative fluorescent PCR carry out the bar of amplified reaction
Part is:
。
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| CN109321682A (en) * | 2018-12-06 | 2019-02-12 | 宁波匠神生物科技有限公司 | A kind of primer pair, primer and probe composition, reagent and kit based on BTNAA system detection canine parvovirus |
| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
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| CN113215310A (en) * | 2021-04-06 | 2021-08-06 | 南京农业大学 | Method for rapidly detecting canine parvovirus through fluorescent quantitative PCR |
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| CN107254555A (en) * | 2017-07-25 | 2017-10-17 | 中国农业科学院特产研究所 | For detecting the primer pair of different subtype canine parvovirus and the combination product of probe |
| CN109321682A (en) * | 2018-12-06 | 2019-02-12 | 宁波匠神生物科技有限公司 | A kind of primer pair, primer and probe composition, reagent and kit based on BTNAA system detection canine parvovirus |
| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
| CN110484656A (en) * | 2019-09-03 | 2019-11-22 | 深圳市慧思基因科技有限公司 | A kind of detection method of canine parvovirus drying type real-time fluorescence PCR detection reagent |
| CN111500793A (en) * | 2020-06-22 | 2020-08-07 | 河北三狮生物科技有限公司 | Detection primer and kit for canine parvovirus and use method of detection primer and kit |
| CN112458205A (en) * | 2020-11-18 | 2021-03-09 | 东莞源和生物科技有限公司 | Reaction solution, primer pair and kit for detecting nucleic acid of feline pestivirus |
| CN113215310A (en) * | 2021-04-06 | 2021-08-06 | 南京农业大学 | Method for rapidly detecting canine parvovirus through fluorescent quantitative PCR |
| CN113215310B (en) * | 2021-04-06 | 2024-06-11 | 南京农业大学 | Method for rapidly detecting canine parvovirus through fluorescent quantitative PCR |
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