CN106435027A - General type PCR (polymerase chain reaction) detection primer, kit and detection method for canine bocavirus - Google Patents
General type PCR (polymerase chain reaction) detection primer, kit and detection method for canine bocavirus Download PDFInfo
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- CN106435027A CN106435027A CN201610925711.9A CN201610925711A CN106435027A CN 106435027 A CN106435027 A CN 106435027A CN 201610925711 A CN201610925711 A CN 201610925711A CN 106435027 A CN106435027 A CN 106435027A
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Abstract
The invention discloses a general type PCR (polymerase chain reaction) detection primer, kit and detection method for a canine bocavirus. The prepared general type PCR detection kit for a canine bocavirus has the advantages of strong specificity, high sensitivity, time saving and labor saving. The method disclosed by the invention is a rapid detection method for diagnosis of a canine bocavirus, fills up the blank of the canine bocavirus diagnosis method in China, and provides corresponding technical support and help for diagnosis, investigation, prevention, control and purification of the canine bocavirus. The method is not only used for disease diagnosis and detection of pet dogs, but also used for detection of wild dogs, meat dogs, experimental dogs and the like.
Description
Technical field
Present invention relates particularly to a kind of universal PCR detection primer of dog bocavirus, test kit and detection method.
Background technology
Bocavirus（Bocavirus）It is a genus of Parvoviridae parvovirus subfamily, be sub-thread wire no cyst membrane
DNA viruses, Genome Size is in 5.4kb.Early 1960s find in cows first.In recent years, with virus
Grand genome and the fast development of high throughput sequencing technologies, human bocavirus, pig bocavirus, dog bocavirus, chimpanzee are won
Card virus, sea lion bocavirus etc. are found in front and back.However, on hereditism, the genetic evolution relation between them farther out,
Genomic homology is less than 60%.Additionally, do not find yet bocavirus exist across kind propagate the fact.
Current research shows, dog bocavirus（Canine Bocavirus, CBoV）Diarrhoea and respiratory tract disease with dog
Disease has certain dependency.More in the case of the area report such as North America, Europe, Korea S, in China, in addition to Hong Kong,
Seldom there is the Case report of dog bocavirus Continental Area.Existing document shows, dog bocavirus Isolation and culture difficulty is relatively
Greatly, and, dog bocavirus genotype is many, has dog bocavirus 1 type（CBoV1）, dog bocavirus 2 type（CBoV2）Win with dog
Block viral 3 types（CBoV3）, between genotype, homology is relatively low.So research and development dog bocavirus PCR universal testing kit is to this disease
Quickly make a definite diagnosis and have great significance.
Content of the invention
It is an object of the invention to carrying out design of primers, sequence alignment with methods such as molecular biology, bioinformatics
And reaction system optimization, set up dog bocavirus versatility detection kit and detection method.This test kit wins card disease for dog
Poison diagnosis provides a kind of method for quick, has filled up the blank of domestic dog bocavirus diagnostic method, has been dog bocavirus
Diagnosis, investigation, prevention and control and purify certain technical support and help are provided.
The technical solution used in the present invention is：
A kind of universal PCR detection primer of dog bocavirus, its nucleotide sequence is as follows：
CBoV-F: AARAGRAARCTYTATTTTGC（SEQ ID NO:1）；
CBoV-R: TGCCAGTCTTGWGGHGARAA（SEQ ID NO:2）.
A kind of Universal PCR detection kit of dog bocavirus, this test kit contains primer described above.
A kind of universal PCR detection method of dog bocavirus, comprises the following steps：
1) extract viral nucleic acid from sample；
2) with nucleic acid as template, carry out pcr amplification reaction with above-mentioned primer pair CBoV-F and CBoV-R and obtain amplified production；
3) pcr amplification product is entered row agarose gel electrophoresis analysis, observed result under gel imaging system, determine virus type
Type.
Preferably, step 2）Middle pcr amplification reaction system is：2 × Taq PCR Master Mix 12.5 μ L, ddH2O
8.5 μ l, DNA profiling 3 μ L, primer CBoV-F 0.5 μ L, primer CBoV-R 0.5 μ L, cumulative volume is 25 L.
Preferably, step 2）Middle PCR response procedures：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72
DEG C extend 30s, altogether 40 circulation；72 DEG C extend 10min eventually.
The invention has the beneficial effects as follows：
The universal PCR detection primer of the dog bocavirus of the present invention, test kit and detection method, are used not only for pet dog
Medical diagnosis on disease and detection, the detection of wild dog, meat dog, experimental dog etc. can also be applied to.
Dog bocavirus Universal PCR detection kit prepared by the present invention, has high specificity, sensitivity height, saves
The advantages of Shi Shengli.
PCR detection method of the present invention provides a kind of method for quick for the diagnosis of dog bocavirus, fills up domestic dog and wins
The blank of card viral diagnosis method, provides certain technical support and side for the diagnosis of dog bocavirus, investigation, prevention and control and purification
Help.
Brief description
Fig. 1 is dog bocavirus Universal PCR detection kit specific test result；Swimming lane M：DNA Marker
2000；Swimming lane 1：Canine distemper virus；Swimming lane 2：Canine Parvovirus；Swimming lane 3：Canine infectious hepatitis viruss；Swimming lane 4：Dog parainfluenza
Virus；Swimming lane 5：Feline distemper virus；Swimming lane 6：Dog bocavirus.
Fig. 2 is dog bocavirus Universal PCR detection kit sensitivity testss result；Wherein swimming lane M：DNA Marker
2000；Swimming lane 1-11 is respectively the 10 of positive plasmid0Again, 101Again, 102Again, 103Again, 104Again, 105Again, 106Again, 107Again, 108
Again, 109Again, 1010Times doubling dilution result.
Specific embodiment
Material and method：
Dog bocavirus positive recombinant plasmid, Canine Parvovirus（CPV）, canine distemper virus（CDV）, canine infectious hepatitis viruss
（ICHV）, canine parainfluenza viruses（CPIV）, feline distemper virus（FDV）Cell separation supernatant etc. is moved by Guangdong Academy of Agricultural Sciences
Thing health research is prepared to be preserved.Additionally, dog bocavirus positive strain is presented by Hong Kong University professor Liu Jiapei.
Main agents：
DNA extraction kit, PCR reagent are purchased from Beijing Tiangeng company, and DNA Marker 2000 is purchased from Takara company.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1 PCR primer designs
Design the primer of amplification dog bocavirus conserved genetic sequences according to dog bocavirus 1 type, 2 types and 3 type gene orders
To CBoV-F and CBoV-R, its base sequence is as follows.
CBoV-F: AARAGRAARCTYTATTTTGC（SEQ ID NO:1）；
CBoV-R: TGCCAGTCTTGWGGHGARAA（SEQ ID NO:2）.
Embodiment 2 viral DNA extracts and pcr analysis
1）The extraction of DNA
Carry out viral DNA extraction according to DNA extraction kit description.
2）Dog bocavirus PCR detection kit
With dog bocavirus（Dog bocavirus 2 type）DNA is the template of PCR, sets up dog bocavirus PCR detection kit.PCR
Reaction system is：2 × Taq PCR Master Mix 12.5 μ L, ddH2O 8.5 μ l, DNA profiling 3 μ L, primer CBoV-F
0.5 μ L, primer CBoV-R 0.5 μ L, cumulative volume is 25 L.
PCR response procedures：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, altogether 40
Individual circulation；72 DEG C extend 10min eventually.
Result is observed：Take 10 μ L PCR primer to carry out 1.5% agarose gel electrophoresiies analysis, see under gel imaging system
Examine result.If there is length for 400bp about target stripe, then be judged to dog bocavirus.
Embodiment 3 specific test
The reaction system being related to the universal PCR kit having optimized in embodiment 2 and condition, thin to dog bocavirus, dog
Small viruses, canine distemper virus, canine infectious hepatitis viruss, canine parainfluenza viruses, feline distemper virus etc. enters performing PCR amplification, to examine
Survey the specificity of this test kit.Result is shown in Fig. 1.
Fig. 1 Gel electrophoresis results show, only expected size in dog bocavirus positive recombinant plasmid（400bp about）
Amplification purpose band, and Canine Parvovirus, canine distemper virus, canine infectious hepatitis viruss, canine parainfluenza viruses, cat distemper calentura
Poison etc. does not assume band, shows that this test kit has preferable specificity.
Embodiment 4 sensitivity testss
Selection dog bocavirus positive recombinant plasmid is template（Concentration is 480.62ng/ μ L), carry out doubling dilution（It is respectively
100Again, 101Again, 102Again, 103Again, 104Again, 105Again, 106Again, 107Again, 108Again, 109Again, 1010Dilute again）, to measure this
The sensitivity of method.Result is shown in Fig. 2.
Fig. 2 result shows：The result of swimming lane 6 is highest extension rate 105, that is, optimize after test kit sensitivity be
4.8062pg/μL.
Embodiment 5 clinical sample detects
Gather clinical 425 parts of dog fecal sample from Guangzhou surrounding area, enter performing PCR detection.
After testing, there is 1 part of dog bocavirus positive sample in 425 parts of clinical samples, further through nucleic acid sequencing, determining should
Sample infects dog bocavirus 2 type（CBoV2）.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment
Limit, other any spirit without departing from the present invention and the change made under principle, modification, replacement, combine, simplify,
All should be equivalent substitute mode, be included within protection scope of the present invention.
SEQUENCE LISTING
<110>Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
<120>A kind of universal PCR detection primer of dog bocavirus, test kit and detection method
<130> CBoV2
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial primer
<400> 1
aaragraarc tytattttgc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial primer
<400> 2
tgccagtctt gwgghgaraa 20
Claims (5)
1. the universal PCR detection primer of a kind of dog bocavirus, its nucleotide sequence is as follows：
CBoV-F: AARAGRAARCTYTATTTTGC（SEQ ID NO:1）；
CBoV-R: TGCCAGTCTTGWGGHGARAA（SEQ ID NO:2）.
2. a kind of Universal PCR detection kit of dog bocavirus it is characterised in that：This test kit contains claim 1 institute
The primer stated.
3. a kind of universal PCR detection method of dog bocavirus is it is characterised in that comprise the following steps：
1）Viral nucleic acid is extracted from sample；
2）With nucleic acid as template, carry out pcr amplification reaction with primer pair CBoV-F described in claim 1 and CBoV-R and obtain expansion
Volume increase thing；
3）Pcr amplification product is entered row agarose gel electrophoresis analysis, observed result under gel imaging system, determines virus type
Type.
4. PCR detection method according to claim 3 is it is characterised in that step 2）Middle pcr amplification reaction system is：2×
Taq PCR Master Mix 12.5 μ L, ddH2O 8.5 μ l, DNA profiling 3 μ L, primer CBoV-F 0.5 μ L, primer CBoV-R
0.5 μ L, cumulative volume is 25 L.
5. PCR detection method according to claim 3 is it is characterised in that step 2）Middle PCR response procedures：95 DEG C of pre- changes
Property 5min；95 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 circulations altogether；72 DEG C extend 10min eventually.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755176A (en) * | 2016-04-22 | 2016-07-13 | 浙江理工大学 | Real-time fluorescent quantitative PCR detecting and typing diagnosis kit for pig bocavirus |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105755176A (en) * | 2016-04-22 | 2016-07-13 | 浙江理工大学 | Real-time fluorescent quantitative PCR detecting and typing diagnosis kit for pig bocavirus |
Non-Patent Citations (3)
| Title |
|---|
| DONGHUA GUO ET AL: "Epidemiological investigation reveals genetic diversity and high co-infection rate of canine bocavirus strains circulating in Heilongjiang province, Northeast China", 《RESEARCH IN VETERINARY SCIENCE》 * |
| S.-L.ZHAI ET AL: "First complete genome sequence of canine bocavirus 2 in mainland China", 《NEW MICROBE AND NEW INFECT》 * |
| 陈意伟等: "犬博卡病毒PCR检测方法的建立及其应用", 《安徽农业科学》 * |
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