CN108359743A - A kind of HRM primer group for differentiating FPV and CPV, the kit containing the primer sets and its application - Google Patents
A kind of HRM primer group for differentiating FPV and CPV, the kit containing the primer sets and its application Download PDFInfo
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Abstract
The invention discloses a kind of HRM primer group for differentiating canine parvovirus (CPV) and feline panleucopenia virus (FPV), the kit containing the primer sets and its application.The primer sets are made of sense primer and downstream primer, and the nucleotide sequence of the sense primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2.In addition, the invention also provides a kind of HRM assay kits containing the primer sets.It is demonstrated experimentally that there is good sensibility and specificity using kit detection FPV and CPV, and it is compared with experiments such as virus purification, determined dna sequences, there is very high coincidence rate, relative specificity and sensibility.The it is proposed of the present invention provides effective technology means for the prevention of canine parvovirus (CPV) and feline panleucopenia virus (FPV) disease.
Description
Technical field
The present invention relates to one kind be used for viral diagnosis primer, the kit containing the primer and its application, more particularly to one
HRM primer of the kind for differentiating FPV and CPV, the kit containing the primer and its application.The invention belongs to viral diagnosis and examine
Disconnected technical field.
Background technology
Parvovirus (Parvoviridae) is sub-thread, linear DNA virus, and average gene group size is 5000nt.According to
International Commission on Virus Classification's newest division in 2016, parvovirus are divided into the parvovirus subfamily that can infect mammal
With the densovirus subfamily of infected insect.Canine parvovirus (Canine parvovirus, CPV) and feline panleucopenia virus
(Feline parvovirus, FPV) belongs to parvovirus subfamily, canine parvovirus belongs to member.
CPV and FPV is the main infection pathogen of domestic dog, cat and wild flesh-eater, genome similarity
It is 98% or more.Eighties of last century the seventies are for the first time from isolated CPV in enteritis dog excrement is suffered from, then rapidly in the whole world
It propagates.Original CPV-2 type constant evolutions are gradually replaced by new hypotype (CPV-2a, CPV-2b, CPV-2c).Research report
Road, CPV are that the host of FPV migrates strain, are the results for adapting to new host.CPV ratios FPV has faster variation rate, they
Respectively with annual 1.7 × 10-4With 9.4 × 10-5The rate variation of a mononucleotide site variation.
Recently, 80% illness cat all infects CPV in Vietnam and TaiWan, China report, in contrast to this in the receiving in the U.S.
In the collecting post of the cat adopt and the mixing raising of dog cat cat body in find that CPV infection rates are 32.5% and 33.9%.Except this
Except, Battilani M et al. report the case that a cat is infected by CPV and FPV simultaneously.However, now in addition to DNA sequencing
Outside, the method for CPV and FPV classification diagnosis is only distinguished using the hemagglutination-inhibition test of monoclonal antibody.Therefore one new
Diagnostic method for after parvovirus infections it is timely treatment be extremely urgent.
With the appearance of saturated fluorescence dyestuff, qPCR instrument can collect more small-scale change in fluorescence, and data can profit
It is directly analyzed with software, high-resolution solubility curve (HRM) method is becoming one quickly, effectively detects the new of clinical sample
Method.Research shows that HRM has been used in the successful discriminating of CPV different subtypes.Therefore, it is an object of the invention to establish one
It is a to reduce time loss, easy to operate, data analysis, while diagnosing and distinguishing the high-resolution of CPV infection and FPV infection
Rate solubility curve method.
Invention content
An object of the present invention is to provide a kind of HRM primer group for differentiating FPV and CPV；
The second object of the present invention is to provide a kind of HRM assay kits for differentiating FPV and CPV；
The third object of the present invention be establish it is a kind of for diagnose and distinguish CPV infection and FPV infection high-resolution it is molten
Solution curve method.
In order to achieve the above object, present invention employs following technological means：
Inventor downloads all canine parvovirus and feline panleucopenia virus whole genome sequence on GenBank, leads to
It crosses MEGA7 and compares analysis, find and there are stable base difference genetic fragment (Reference strains：CPV Laika-1993 are logged in
Number：JN033694.1), a pair of of qPCR primer sets (F2/R2) of CPV and FPV are distinguished in design, and the primer sets are drawn by upstream
Object and downstream primer composition, the nucleotide sequence of the sense primer as shown in SEQ ID NO.1, the downstream primer
Nucleotide sequence is as shown in SEQ ID NO.2.
Further, the invention also provides the primer sets to prepare detection or differentiate that feline panleucopenia virus and dog are tiny
Purposes in the reagent of virus infection.
Further, the invention also provides one kind for differentiating feline panleucopenia virus (FPV) and canine parvovirus
(CPV) HRM assay kits, the kit include primer sets of the present invention.
Wherein, it is preferred that the kit further includes the PCR reaction solution containing saturated fluorescence dyestuff, positive criteria matter
Grain and sterile water.
Wherein, it is preferred that the positive criteria plasmid is prepared in accordance with the following methods：Feline panleucopenia virus is extracted respectively
With canine parvovirus DNA, using the DNA of extraction as template, with sequence shown in SEQ ID NO.3 and SEQ ID NO.4 be draw
Object carries out PCR amplification；It is connect with carrier to get positive criteria plasmid after PCR product is purified.
Wherein, it is preferred that the carrier is pEASY-T5zero.
When feline panleucopenia virus and Canine parvovirus infection are detected or differentiated using kit of the present invention, according to following
Step carries out：
(1) total DNA of sample to be tested is extracted
(2) PCR-HRM is detected
The DNA obtained using step (2) carries out quantitative fluorescent PCR expansion as template, using primer pair described in claim 1
Increase, quantitative fluorescent PCR system is：
PCR-HRM response procedures are：50 DEG C of 2min, 95 DEG C of pre-degeneration 10min；95 DEG C are denaturalized 15s, and 58 DEG C of annealing 40s are followed
Ring 35 times；95 DEG C of denaturation 15s, 60 DEG C of 1min to 95 DEG C of 15s carry out solubility curve analysis with the rate of dissolution of 0.025 DEG C/s.
Compared to the prior art, the beneficial effects of the present invention are：The present invention by compare CPV and FPV nucleic acid sequence,
Conservative difference site is found out, HRM primer is designed, real-time PCR amplifications is carried out, utilizes Applied
High Resolution Melt Software v3.1 softwares carry out Data Analysis Services, finally establish for detecting or reflecting
The HRM methods of other feline panleucopenia virus and canine parvovirus.It is demonstrated experimentally that this method has good sensibility and specificity, and
And compared with being compared with experiments such as virus purification, determined dna sequences, as a result, it has been found that, HRM methods meet compared with DNA sequencing
Rate, relative specificity and sensibility are respectively 96.25%, 91.43%, 100%；HRM methods with virus purification test compared with,
Coincidence rate, relative specificity and sensibility are respectively 85%, 72.72%, 100%.The it is proposed of the present invention is canine parvovirus
(CPV) and the prevention of feline panleucopenia virus (FPV) disease provides effective technology means.
Description of the drawings
Fig. 1 is the qualification result of positive plasmid；
M,DL2000DNA Marker；1, negative control；2,pEASY-FPV-H；3, pEASY-CPV-2a, 4, it is positive right
According to；
Fig. 2 is the analysis result of HRM methods of the present invention；
Fig. 3 is the specific test result of HRM methods of the present invention；
Fig. 4 is the sensitivity tests result of HRM methods of the present invention.
Wherein Fig. 4 A are calibration solubility curve；Fig. 4 B, 4C are respectively CPV and FPV amplification standard curves.
Specific implementation mode
The present invention is further described with reference to specific embodiments and the drawings, the advantages and features of the present invention will be with
Description and it is apparent.But examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Art technology
Personnel should be understood that without departing from the spirit and scope of the invention can be to the details and form of technical solution of the present invention
It modifies or replaces, but these modifications and replacement are each fallen in protection scope of the present invention.
1 design of primers of embodiment
All canine parvovirus and feline panleucopenia virus whole genome sequence are downloaded on GenBank, are compared by MEGA7
Analysis is found and there are stable base difference genetic fragment (Reference strains：CPV Laika-1993, accession number：
JN033694.1), a pair of of qPCR primers (F2/R2) of CPV and FPV are distinguished in design.And design pair of primers (F1/R1) amplification packet
It includes genetic fragment structure CPV including qPCR amplified fragments and FPV standard plasmids, primer sequence is as shown in table 1.
1 primer sequence of table
The foundation of embodiment 2 high-resolution solubility curve (HRM) method
1, the structure of standard plasmid
200ul CPV virus stock solution useds and FPV virus stock solution useds are taken respectively, extract viral DNA.Operating procedure is by TAKARA's
The operating instruction of MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 carries out.The DNA extracted with kit
For template, with F1/R1 (table 1) for primer, reference TaqTM(Ex TaqTM Version 2.0plus dye)(Takara,
RR902A) operating instruction carries out PCR amplification, and PCR system (50ul) is as shown in table 2.
2 PCR system of table (50ul)
Amplification response procedures are in advance：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 1min, 72 DEG C extend
2min；Cycle 35 times；72 DEG C extend 10min eventually.
3% gel is prepared with the buffer solution (1 × TAE) diluted.5ul PCR products are taken to be added in gel pore, electrophoresis is seen
Examine result.Remaining PCR product referencePCR Purfication Kit operation instructions are purified.It will after purification
DNA be attached conversion, by PCR and sequencing identification positive monoclonal plasmid be named as respectively pEASY-FPV-H and
PEASY-CPV-2a, and be stored in -80 DEG C it is spare.Connection illustrates to carry out with reference to pEASY-T5zero carrier operations.
2, qPCR amplifications and HRM analyses
Respectively using pEASY-FPV-H and pEASY-CPV-2a and the two in equal volume than mixture as template, with primers F 2/
R2 (table 1), in ABI QuantStudioTMOn Design fluorescence quantitative PCR instruments, carried out according to the system of best fluorescent quantitation
Amplification, it is as shown in table 3 that HRM tests reaction system.
Table 3 HRM experiment reaction systems (30ul)
PCR-HRM response procedures are：50 DEG C of 2min, 95 DEG C of pre-degeneration 10min；95 DEG C are denaturalized 15s, and 58 DEG C of annealing 40s are followed
Ring 35 times；95 DEG C of denaturation 15s, 60 DEG C of 1min to 95 DEG C of 15s carry out solubility curve analysis with the rate of dissolution of 0.025 DEG C/s.HRM
Test result AppliedHigh Resolution Melt Software v3.1 are analyzed.The software
The similarity of melting curve can be relied on to sort out unknown sample automatically.The results are shown in Figure 2.
The result shows that the HRM methods that the present invention establishes can distinguish CPV, FPV and its mixture.
3, specific test
With CPV and FPV and other dog sources virus：Hepatitis infectiosa canis virus CAV-2C, canine coronavirus CCV HB16-2, hundstaupe pyreticosis
The DNA or cDNA of malicious CDV3 is template, and fluorescent quantitation is carried out in the case where optimizing reaction condition, identifies its specificity.
The results are shown in Figure 3, should the result shows that, which can amplify CPV-2,2a, 2b, 2c types and FPV, and CCV,
The virus such as CAV and CDV shows this method high specificity without amplification curve.
4, sensitivity tests
The standard plasmid pEASY-FPV-H and pEASY-CPV-2a of CPV and FPV is serially diluted into 9 again with distilled water 10
A gradient, make its final concentration of 4.2 × 108Copies/ul~4.2 × 100copies/ul.As template, in ABI
It is expanded on QuantStudioTM Design fluorescence quantitative PCR instruments, and carries out HRM analyses.Initial amplification is less than 35Ct quilts
It is considered positive amplification.The lowest detection copy number of HRM methods is 4.2copies/ μ l, as a result as shown in Figure 4 A.In addition to this,
After CPV and FPV amplifications, standard curve is the results show that 4.2 × 108Copies/ul~4.2 × 100Have between copies/ul
Good linear relationship, normal equation are respectively Ct=-3.441lg copiese+39.988, Ct=-3.377lg
Copiese+39.593 (Fig. 4 B and Fig. 4 C).
3 clinical application of embodiment
80 parts of suspected infection canine parvovirus or feline panleucopenia virus are acquired from Beijing, Shenyang, Shijiazhuang and Changchun and other places
Clinical sample carries out clinical detection according to the HRM methods that embodiment 2 is established, at the same with the examinations such as virus purification, determined dna sequence
It tests to be compared and compare.HRM analysis results are found, share 42 parts of CPV positives, 6 parts of FPV positives.45 parts of sample quilts
Success is sequenced, CPV the and FPV positives are respectively 39 parts, 6 parts.Virus purification experiments are carried out to 80 parts of samples, obtain 32 plants of CPV, 4
Strain FPV.HRM methods are compared with DNA is sequenced, and coincidence rate, relative specificity and sensibility are respectively 96.25%, 91.43%,
100%；HRM methods are compared with virus purification is tested, and coincidence rate, relative specificity and sensibility are respectively 85%, 72.72%,
100%.
Sequence table
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<120>A kind of HRM primer group for differentiating FPV and CPV, the kit containing the primer sets and its application
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Claims (7)
1. HRM primer group of the one kind for differentiating feline panleucopenia virus (FPV) and canine parvovirus (CPV), which is characterized in that by upper
Primer and downstream primer composition are swum, the nucleotide sequence of the sense primer as shown in SEQ ID NO.1, draw by the downstream
The nucleotide sequence of object is as shown in SEQ ID NO.2.
2. primer sets described in claim 1 are in preparing detection or differentiating the reagent of feline panleucopenia virus and Canine parvovirus infection
Purposes.
3. HRM assay kit of the one kind for differentiating feline panleucopenia virus (FPV) and canine parvovirus (CPV), which is characterized in that
The kit includes primer sets described in claim 1.
4. kit as claimed in claim 3, which is characterized in that the kit further includes containing saturated fluorescence dyestuff
PCR reaction solution, positive criteria plasmid and sterile water.
5. kit as claimed in claim 3, which is characterized in that the positive criteria plasmid is prepared into accordance with the following methods
It arrives：Feline panleucopenia virus and canine parvovirus DNA are extracted respectively, using the DNA of extraction as template, with SEQ ID NO.3 and SEQ
Sequence shown in ID NO.4 is primer, carries out PCR amplification；It is connect with carrier to get positive criteria matter after PCR product is purified
Grain.
6. kit as claimed in claim 5, which is characterized in that the carrier is pEASY-T5zero.
7. such as claim 3-6 any one of them kits, which is characterized in that for detecting or differentiating feline panleucopenia virus and dog
When parvovirus infections, follow the steps below：
(1) total DNA of sample to be tested is extracted
(2) PCR-HRM is detected
The DNA obtained using step (2) carries out fluorescent quantitative PCR as template, using primer pair described in claim 1, glimmering
Fluorescent Quantitative PCR system is：
PCR-HRM response procedures are：50 DEG C of 2min, 95 DEG C of pre-degeneration 10min；95 DEG C of denaturation 15s, 58 DEG C of annealing 40s recycle 35
It is secondary；95 DEG C of denaturation 15s, 60 DEG C of 1min to 95 DEG C of 15s carry out solubility curve analysis with the rate of dissolution of 0.025 DEG C/s.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819735A (en) * | 2018-08-13 | 2020-02-21 | 北京美莱博医学科技有限公司 | Complete set of reagent and kit for identifying canine parvovirus |
| CN111518955A (en) * | 2020-05-15 | 2020-08-11 | 广东省实验动物监测所 | HRM primer pair, kit and method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus |
| CN111518961A (en) * | 2020-06-28 | 2020-08-11 | 鲁东大学 | Primer for gene amplification of feline coronavirus and genotyping method |
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| CN103160614A (en) * | 2011-12-15 | 2013-06-19 | 广州世优生物科技有限公司 | Universal PCR detection kit for cat and dog parvovirus |
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2017
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| CN103160614A (en) * | 2011-12-15 | 2013-06-19 | 广州世优生物科技有限公司 | Universal PCR detection kit for cat and dog parvovirus |
| CN104004857A (en) * | 2014-05-27 | 2014-08-27 | 广州博至生物科技有限公司 | PCR-HRM primer and method for quickly distinguishing canine parvoviruses of different genotypes |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819735A (en) * | 2018-08-13 | 2020-02-21 | 北京美莱博医学科技有限公司 | Complete set of reagent and kit for identifying canine parvovirus |
| CN111518955A (en) * | 2020-05-15 | 2020-08-11 | 广东省实验动物监测所 | HRM primer pair, kit and method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus |
| CN111518961A (en) * | 2020-06-28 | 2020-08-11 | 鲁东大学 | Primer for gene amplification of feline coronavirus and genotyping method |
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