CN103160614A - Universal PCR detection kit for cat and dog parvovirus - Google Patents
Universal PCR detection kit for cat and dog parvovirus Download PDFInfo
- Publication number
- CN103160614A CN103160614A CN 201110421809 CN201110421809A CN103160614A CN 103160614 A CN103160614 A CN 103160614A CN 201110421809 CN201110421809 CN 201110421809 CN 201110421809 A CN201110421809 A CN 201110421809A CN 103160614 A CN103160614 A CN 103160614A
- Authority
- CN
- China
- Prior art keywords
- parvovirus
- cat
- dog
- pcr
- pcr detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a universal PCR detection kit for rapid and high-efficient detection of cat and dog parvovirus. The method comprises designing a pair of primers in accordance with a conservation region of a vp2 gene of the cat and dog parvovirus, performing a PCR reaction by using excrement of detected animals as a template, comparing with negative and positive controls included in the kit, and determining whether the detected animals are infected with the parvovirus. The detection kit is simple, easy to use, high in sensitivity, and good in stability, and can be used for monitoring health conditions of the cat and the dog.
Description
Technical field
The invention belongs to zoonotic diagnostic techniques, relate to the detection method to the parvovirus of cat and dog.
The invention provides a kind of universal PCR test kit that detects the cat and dog parvovirus.Content of the present invention relates to and a pair ofly can be simultaneously detects the PCR primer of cat and dog parvovirus with rice, and a cover has comprised the PCR detection kit that PCR reaction mix (containing primer), positive control, negative control, tetrabromophenol sulfonphthalein sample-loading buffer form and detected accordingly schedule of operation.
Background technology
Feline panleucopenia virus (felinepanleukopenia virus, FPV) claim again Feline Panleukopenia Virus, feline infectious enteritis virus or feline panleukopenia virus, is one of main pathogen of harm feline; And canine parvovirus (canine parvovirus, CPV) is to cause dog acute hemorrhagic gastro-enteritis and the myocarditic main pathogen of pup.The clinical symptom of animal of catching an illness is useless absolutely for high heat, vomiting, appetite, fervescence, serious dehydration, spiritual depressed etc.Dissect finding has typical enteritis symptom, as the enteron aisle oedema, hemorrhage, mucous membrane comes off, mesenteric adenophyma is swollen, liver, kidney enlargement and hemostasis etc.Particularly fall ill ight soil, secretory product and the vomitus of animal of infection animal all is with poison, and virus can be propagated by approach such as direct contact or digestive tube, respiratory tracts.
FPV and CPV are single-stranded DNA viruses, and the homology between genome surpasses 98%, and virion has identical structure; Viral genome has 2 open reading frame, encode respectively Nonstructural Protein (NS) and structural protein (VP); 2 kinds of viruses all contain VP1 and VP2 capsid protein, wherein VP2 is main capsid protein, and it has comprised all neutralizing antigenic sites, and its full length gene is 1755bp, 584 amino acid of encoding, the decision of the crucial base of the youngster on the vp2 gene antigenic characteristic and the host range of strain.Difference as the 93rd and 323 key amino acid residue on VP2 albumen in FPV and CPV has determined that there is obvious difference in they on antigenicity.The N end of VP2 albumen and corner structure district Loop 1 thereof and Loop 3 are important B cell antigen epi-position districts, can induce host cell to produce neutralizing antibody, and the variation of these regional amino-acid residues can cause strain to produce antigenic drift.Have result of study to show, the variation of 6 amino acid (80,93,103,323,564,568 amino acids) has only occured in FPV to the evolution of CPV22, and the variation of a few amino acids is also only arranged between each variant of CPV22.This shows, although the base quantity that viral genome changes is few, can cause the antigenicity of virus, pathogenic, cell tropism, host range etc. that noticeable change occurs.
These 2 kinds of viruses of CPV and FPV are closely related on evolving, and a kind of well accepted hypothesis is that FPV is the ancestors of CPV.Although the Relationship Comparison between these 2 kinds of viruses is complicated, CPV especially, many variants have appearred in rice in recent years, and the difference between the difference between these strains and this 2 kinds of viruses has been brought difficulty to genetic evolution research of FPV.Although CPV and FPV are at genetics and biologically in close relations, their evolutionary mechanism is different.The gene time to time change of FPV coding non-structural protein 1 (NS1) and capsid protein 2 (VP2), but VP2 albumen does not change.The VP2 albumen of CPV changes with the variation of its gene.That is, in FPV, mostly the sudden change of NS1 and VP2 is same sense mutation, and the vp2 transgenation of CPV is mainly missense mutation.This explanation is compared with FPV, and CPV is mostly is in the situation that selective pressure is arranged, and constantly produces new mutant strain by antigenic drift, to escape host's immunologic mechanism.A lot of variations have occured in the youngster of CPV after it occurs on antigenicity, host range and blood clotting in the year, the hypotype that has occurred at present comprises CPV22a, CPV22b, CPV22c (a) and CPV22c (b) etc.
Find by the virulence research to CPV, FPV, FPV, CPV22 all can breed in the cat source cell, but only have CPV22 to grow in the dog source cell.The virulence that FPV, CPV will compare heterologous host to origin host's virulence is much better than.And along with the continuous variation of CPV22 hypotype, each hypotype to the infection ability of feline also in continuous enhancing.
At present the detection method of parvovirus mainly contained viral separation and Culture, fluorescent-antibody technique, Electronic Speculum and immunoelectronmicroscopy, blood coagulation tests and blood clotting inhibition experiment, enzyme linked immunological experiment etc.But all there is certain shortcoming and defect in above method.Laboratory condition and professional technique that the requirement of virus separation and Culture is necessary; The technology that Electronic Speculum is relevant needs expensive plant and instrument; Fluorescent-antibody technique only is confined to detect the virus in pathological tissue or cell culture; Enzyme-linked immunosorbent assay is subject to the impact of interfering factors, and susceptibility is relatively poor; Blood coagulation tests and blood clotting suppress experiment need prepare fresh responsive red corpuscle at any time, sometimes also need do blood clotting and suppress the experiment proved.
PCR can hypersensitivity, the rapid detection target gene DNA of high specific.Even also can efficiently detect when containing trace parvovirus DNA in sample.Though have at present the report to the PCR detection method of canine parvovirus, the report of feline panleucopenia virus PCR detection kit is not arranged, also there is no to detect simultaneously the report of the universal test kit of cat and dog parvovirus.
The genome sequence height of CPV and FPV is consistent, therefore can be according to the conservative region design pair of primers of vp2 gene, and specific amplification contains the fragment of vp2 gene hypervariable region.
Summary of the invention
The object of the present invention is to provide a kind of universal PCR test kit and corresponding operating program that detects the cat and dog parvovirus.Utilize that the primer of high degree of specificity can efficiently and accurately from being amplified the purpose fragment the animal excrement of parvovirus infections or secretory product, rapidly and efficiently, recall rate is high, result is accurate, cost is controlled.
Technical essential of the present invention is the design primer, and the purpose fragment of specific amplification provides foundation for clinical diagnosis, and can judge accurately cause of disease by order-checking rice when being necessary.For achieving the above object, the technical solution used in the present invention is as follows:
According to parvovirus VP2 gene conserved regions design pair of primers, primer sequence is PF 5 '-TTCTGTGCCAGTACACTTAC-3 ' and PR, 5 '-CCTGTATCTTGATGTGC-3 '.PCR product length is 477bp, and its sequence is seen the sequence 3 in sequence table.
A kind of PCR detection kit of cat and dog parvovirus comprises: (1) 2 * PCR reaction mix.Contain the PCR reaction buffer in mix, dNTP, primer PF and PR; Archaeal dna polymerase; (2) positive control (recombinant plasmid that contains canine parvovius vp2 gene conserved regions); (3) negative control (Healthy Cats or dog are without the excrement sample filtrate of poisoning the asepticize processing); (4) tetrabromophenol sulfonphthalein sample-loading buffer; (5) 1.5ml centrifuge tube; (6) sampling swab; (7) sample preparation damping fluid.
The use flow process of one this test kit of cover comprises: (1) sample preparation.Scrape with sampling swab the animal excreta sample to be checked (or urine, nasal secretions etc.) that takes a morsel and be placed in the 1.5ml centrifuge tube, add 1ml sample preparation damping fluid, fully vibration makes the excrement sample abandon swab after swab comes off, centrifugal 1 minute of 12000g, get supernatant and put into another sterilization centrifuge tube, boiling water bath ten minutes, cooling rear 12000g centrifugal ten minutes, draw supernatant and change in another cleaning sterile centrifuge tube ,-20 ℃ save backup; (2) PCR.Reaction system is 20 μ l.Add respectively 10 μ l PCR mix in three 0.2ml EP pipes, add 8 μ l aseptic deionized waters, 2 μ l samples or the positive and negative control, the rearmounted PCR instrument of mixing increases.The PCR reaction conditions is: 95 ℃ of denaturation 10min, and 94 ℃ of sex change 30sec, 55 ℃ of annealing 30sec, 72 ℃ are extended 30sec, circulate 30 times.After loop ends, 72 ℃ of polishings extend the end of 10min afterreaction.(3) agarose electrophoretic analysis is identified the PCR product.With sample and positive and negative control electrophoresis evaluation together, after completing, through observing under ultraviolet lamp after ethidium bromide staining, each swimming lane electrophoretic band situation relatively is to judge that sample is as being subjected to the sample product whether with parvovirus with gel.
That uses that universal PCR test kit of the present invention both can quickness and high efficiency detects animal and whether infects parvovirus, and can be by order-checking to determine the cause of disease kind, for the clinical diagnosis treatment facilitate under the condition of necessity.
Description of drawings
Accompanying drawing 1: a doubtful parvovirus infections cat PCR result.The purpose clip size is 477bp (lower same).
The 1st swimming lane, DL2000marker, stripe size (bp) is respectively 2000,1000,750,500,250,100 (lower same) from top to bottom; The 2nd swimming lane, positive control; The 3rd swimming lane, sample P CR result: the 4th swimming lane, negative control.
Accompanying drawing 2: a doubtful parvovirus infections dog PCR result
The 1st swimming lane, DL2000marker; The 2nd swimming lane, positive control; The 3rd swimming lane, sample P CR result; The 4th swimming lane, negative control.
Accompanying drawing 3: a doubtful parvovirus infections dog PCR result
The 1st swimming lane, DL2000marker; The 2nd swimming lane, positive control; The 3rd swimming lane, sample P CR result; The 4th swimming lane, negative control.
Embodiment
The invention will be further described below in conjunction with embodiment, but the present invention is not subjected to the restriction of embodiment.
Molecular biology working method in all embodiment is familiar with by these those skilled in the art, can be with reference to (the laboratory manual such as Sambrook " molecular cloning ", the cold spring port, 1989) reach " fine works molecular biology experiment guide " (work such as U.S./F. Ao Sibai, Yan Ziying etc. translate, Beijing, Science Press, 1998).
Embodiment one
Get one of the ill cat of certain doubtful parvovirus infections, scraping ight soil a little, press test kit and use flow operations, result as shown in Figure 1.Judge that according to result this cat has only infected parvovirus.The detection paper result is also positive, has confirmed the PCR detected result correct.
Embodiment two
Get one of the ill dog of certain doubtful parvovirus infections, the scraping nasal secretions a little, press test kit and use flow operations, result as shown in Figure 2.Judge that according to result this dog has only infected parvovirus.The detection paper result is also positive, has confirmed the PCR detected result correct.
Embodiment three
Get one of the ill dog of certain doubtful parvovirus infections, collect urine a little, press test kit use flow operations, result as shown in Figure 3.Judge that according to result this dog does not only infect parvovirus.The detection paper result is negative, has confirmed the PCR detected result correct.
Claims (3)
1. universal parvovirus PCR detection kit, this test kit comprises: a pair of Auele Specific Primer, PCR reacts mix, positive control dna and negative control.Its feature is universal refers to that this reagent is applicable to the parvovirus PCR detection of cat and dog simultaneously.
2. according to claim 1, specific primer sequence is PF 5 '-TTCTGTGCCAGTACACTTAC-3 ' and PR, 5 '-CCTGTATCTTGATGTGC-3 '.
3. according to claim 1, positive control dna is to contain the recombinant plasmid that length is 477bp cat and dog parvovirus conserved regions DNA sequence dna, and the negative control sample refers to that the nothing of Healthy Cats or dog poisons the excrement sample filtrate that asepticize is processed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110421809 CN103160614A (en) | 2011-12-15 | 2011-12-15 | Universal PCR detection kit for cat and dog parvovirus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110421809 CN103160614A (en) | 2011-12-15 | 2011-12-15 | Universal PCR detection kit for cat and dog parvovirus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103160614A true CN103160614A (en) | 2013-06-19 |
Family
ID=48584148
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110421809 Pending CN103160614A (en) | 2011-12-15 | 2011-12-15 | Universal PCR detection kit for cat and dog parvovirus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103160614A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106888985A (en) * | 2015-12-18 | 2017-06-27 | 英业达科技有限公司 | The outer display information of pet is monitored to judge the system and method for pet health state |
| CN108359743A (en) * | 2017-12-27 | 2018-08-03 | 中国农业科学院特产研究所 | A kind of HRM primer group for differentiating FPV and CPV, the kit containing the primer sets and its application |
| CN109097488A (en) * | 2018-07-09 | 2018-12-28 | 北京市农林科学院 | For synchronizing nucleic acid, method and the kit of five kinds of dog diarrhea virus of detection and identification |
| 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 |
| CN110643744A (en) * | 2019-11-19 | 2020-01-03 | 南京农业大学 | Quantitative detection method for simultaneously detecting three cat susceptible viruses and primer probe combination thereof |
-
2011
- 2011-12-15 CN CN 201110421809 patent/CN103160614A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106888985A (en) * | 2015-12-18 | 2017-06-27 | 英业达科技有限公司 | The outer display information of pet is monitored to judge the system and method for pet health state |
| CN108359743A (en) * | 2017-12-27 | 2018-08-03 | 中国农业科学院特产研究所 | A kind of HRM primer group for differentiating FPV and CPV, the kit containing the primer sets and its application |
| CN109097488A (en) * | 2018-07-09 | 2018-12-28 | 北京市农林科学院 | For synchronizing nucleic acid, method and the kit of five kinds of dog diarrhea virus of detection and identification |
| 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 |
| CN110643744A (en) * | 2019-11-19 | 2020-01-03 | 南京农业大学 | Quantitative detection method for simultaneously detecting three cat susceptible viruses and primer probe combination thereof |
| CN110643744B (en) * | 2019-11-19 | 2022-10-21 | 南京农业大学 | Quantitative detection method for simultaneously detecting three cat susceptible viruses and primer probe combination thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109593893A (en) | African hog cholera virus fluorescent PCR quick detection kit | |
| Greiser-Wilke et al. | Diagnostic methods for detection of Classical swine fever virus—status quo and new developments | |
| US20080280296A1 (en) | Method for detection of foot-and-mouth disease virus with chromatographic strip test | |
| Shin et al. | Comparison of one‐step RT‐PCR and a nested PCR for the detection of canine distemper virus in clinical samples | |
| CN103160614A (en) | Universal PCR detection kit for cat and dog parvovirus | |
| CN104328218A (en) | Nucleic acids of liquid-phase gene chip for synchronously detecting five porcine viruses and detection method thereof | |
| CN101696454A (en) | RT-LAMP primer for visually detecting wild strains of classical swine fever virus | |
| CN105400906A (en) | Triple PCR primer set capable of simultaneously detecting porcine circovirus II, porcine pseudorabies virus and porcine parvovirus and application thereof | |
| CN1840703A (en) | Multiple RT-PCR identification and detection reagent for animal vesicular disease and preparation method and use thereof | |
| Zhu et al. | Rapid and sensitive detection of bovine coronavirus and group a bovine rotavirus from fecal samples by using one-step duplex RT-PCR assay | |
| KR101064866B1 (en) | Primers capable of simultaneous detecting bovine coronavirus, bovine rotavirus and bovine viral diarrhea virus, and simultaneous detection method of diarrhea viruses in ruminants, including cattle, using the same | |
| CN104498623B (en) | Primers for differential diagnosis of porcine epidemic diarrhea virus virulent strain and attenuated strain and detection kit thereof | |
| CN101724712B (en) | Animal insect-borne disease multi-RT-PCR distinguishing and detecting reagent as well as preparation method and application | |
| CN104263839A (en) | LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for brucella | |
| Thuy et al. | First investigation of the prevalence of parvoviruses in slaughterhouse pigs and genomic characterization of ungulate copiparvovirus 2 in Vietnam | |
| Siafakas et al. | Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis | |
| CN102154512A (en) | Fluorescence quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for rabbit hemorrhagic disease virus | |
| CN103757137B (en) | Common primer nucleic acid amplification method for detecting three pig viruses synchronously and kit | |
| RU2766344C1 (en) | Method for detection and identification of coronaviruses in cattle | |
| CN103789430A (en) | Kit for rapid detection of eperythrozoon, leptospira and toxoplasma in blood and application of kit | |
| CN103131797A (en) | Bocavirus real-time fluorescence PCR detection kit and application thereof | |
| CN106521029A (en) | Multiple RT-PCR detection kit for identifying PRRSV (Porcine Reproductive and Respiratory Syndrome) | |
| CN102776294B (en) | Quick detecting kit for C-type foot and mouth disease virus | |
| CN106188249A (en) | For detecting the antigen of PEDV variant antibody and method and test kit | |
| CN111500793A (en) | Detection primer and kit for canine parvovirus and use method of detection primer and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| DD01 | Delivery of document by public notice |
Addressee: Withyou Biotechnology Co., Ltd. Guo Lei Document name: Notification of Passing Preliminary Examination of the Application for Invention |
|
| C06 | Publication | ||
| PB01 | Publication | ||
| DD01 | Delivery of document by public notice |
Addressee: Withyou Biotechnology Co., Ltd. Document name: Notification of Publication of the Application for Invention |
|
| DD01 | Delivery of document by public notice |
Addressee: Withyou Biotechnology Co., Ltd. Document name: Notification of before Expiration of Request of Examination as to Substance |
|
| DD01 | Delivery of document by public notice |
Addressee: Withyou Biotechnology Co., Ltd. Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130619 |