CN106435027B - A kind of universal PCR detection primer, kit and the detection method of dog bocavirus - Google Patents
A kind of universal PCR detection primer, kit and the detection method of dog bocavirus Download PDFInfo
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- CN106435027B CN106435027B CN201610925711.9A CN201610925711A CN106435027B CN 106435027 B CN106435027 B CN 106435027B CN 201610925711 A CN201610925711 A CN 201610925711A CN 106435027 B CN106435027 B CN 106435027B
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Abstract
The invention discloses universal PCR detection primer, kit and the detection methods of a kind of dog bocavirus.Dog bocavirus Universal PCR detection kit prepared by the present invention has the advantages that high specificity, sensibility are high, time saving and energy saving.The method of the present invention provides a kind of rapid detection method for the diagnosis of dog bocavirus, has filled up the blank of domestic dog bocavirus diagnostic method, provides corresponding technical support for the diagnosis of dog bocavirus, investigation, prevention and control and purification and helps.The method of the present invention is used not only for the medical diagnosis on disease and detection of pet dog, can also be applied to the detection of wild dog, meat dog, experimental dog etc..
Description
Technical field
Present invention relates particularly to universal PCR detection primer, kit and the detection methods of a kind of dog bocavirus.
Background technique
Bocavirus (Bocavirus) is a category of Parvoviridae parvovirus subfamily, is sub-thread threadiness without cyst membrane
DNA virus, Genome Size is in 5.4kb or so.Early 1960s find in cows for the first time.In recent years, with virus
The fast development of macro genome and high throughput sequencing technologies, human bocavirus, pig bocavirus, dog bocavirus, chimpanzee are rich
Card virus, sea lion bocavirus etc. are found by front and back.However, on science of heredity, the genetic evolution relationship between them farther out,
Genomic homology is lower than 60%.In addition, do not find yet bocavirus exist across kind propagate the fact.
It is current studies have shown that the diarrhea and respiratory tract disease of dog bocavirus (Canine Bocavirus, CBoV) and dog
Disease has certain correlation.It is more in the case of the area report such as North America, Europe, Korea S, in China, other than Hong Kong,
The Case report of the few dog bocavirus in Continental Area.Existing literature show dog bocavirus Isolation and culture difficulty compared with
Greatly, moreover, dog bocavirus genotype is more, there are 1 type of dog bocavirus (CBoV1), 2 type of dog bocavirus (CBoV2) and dog rich
Block viral 3 types (CBoV3), homology is lower between genotype.So research and development dog bocavirus PCR universal testing kit is to the disease
Quickly make a definite diagnosis and have great significance.
Summary of the invention
It is an object of the invention to carry out design of primers, sequence alignment with the methods of molecular biology, bioinformatics
And reaction system optimization, set up dog bocavirus versatility detection kit and detection method.The kit is the rich card disease of dog
Poison diagnosis provides a kind of rapid detection method, has filled up the blank of domestic dog bocavirus diagnostic method, is dog bocavirus
Diagnosis, investigation, prevention and control and purification certain technical support and help is provided.
The technical solution used in the present invention is:
A kind of universal PCR detection primer of dog bocavirus, nucleotide sequence are 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, the kit contain primer described above.
A kind of universal PCR detection method of dog bocavirus, including the following steps:
1) viral nucleic acid is extracted from sample；
2) using nucleic acid as template, pcr amplification reaction is carried out with above-mentioned primer pair CBoV-F and CBoV-R and obtains amplified production；
3) pcr amplification product is subjected to agarose gel electrophoresis analysis, the observation under gel imaging system is as a result, determine disease
Malicious type.
Preferably, pcr amplification reaction system in step 2 are as follows: 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 of 0.5 μ L, primer CBoV-R, total volume are 25 μ L.
Preferably, PCR response procedures in step 2: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72
DEG C extend 30s, amount to 40 circulation；72 DEG C extend 10min eventually.
The beneficial effects of the present invention are:
Universal PCR detection primer, kit and the detection method of dog bocavirus of the invention, are used not only for doting on
The medical diagnosis on disease and detection of object dog can also be applied to the detection of wild dog, meat dog, experimental dog etc..
Dog bocavirus Universal PCR detection kit prepared by the present invention has high specificity, sensibility height, saves
The advantages that Shi Shengli.
PCR detection method of the present invention provides a kind of rapid detection method for the diagnosis of dog bocavirus, and it is rich to fill up domestic dog
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
It helps.
Detailed description of the invention
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 virus；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 tests 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), dog infectious hepatitis
Poison (ICHV), canine parainfluenza virus (CPIV), feline distemper virus (FDV) cell separate supernatant etc. by Guangdong Academy of Agricultural Sciences
Animal health research is prepared to be saved.In addition, 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.
The present invention is further illustrated combined with specific embodiments below, and however, it is not limited to this.
The design of 1 PCR primer of embodiment
Amplification dog bocavirus conserved genetic sequences are designed according to 1 type of dog bocavirus, 2 types and 3 type gene orders
Primer pair CBoV-F and CBoV-R, base sequence are as follows.
CBoV-F:AARAGRAARCTYTATTTTGC(SEQ ID NO:1)；
CBoV-R:TGCCAGTCTTGWGGHGARAA(SEQ ID NO:2).
2 viral DNA of embodiment extracts and PCR analysis
1) extraction of DNA
Viral DNA extraction is carried out according to DNA extraction kit specification.
2) dog bocavirus PCR detection kit
The template for being PCR with dog bocavirus (2 type of dog bocavirus) DNA, establishes dog bocavirus PCR detection reagent
Box.PCR reaction system are as follows: 2 × Taq PCR Master Mix 12.5 μ L, ddH28.5 μ l of O, 3 μ L of DNA profiling, primer
CBoV-F 0.5 μ L of 0.5 μ L, primer CBoV-R, total volume are 25 μ L.
PCR response procedures: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s amount to 40
A circulation；72 DEG C extend 10min eventually.
As a result it observes: taking 10 μ L PCR products to carry out 1.5% agarose gel electrophoresis analysis, seen under gel imaging system
Examine result.The target stripe for being 400bp or so if there is length, then be determined as dog bocavirus.
3 specific test of embodiment
With reaction system and condition that the universal PCR kit optimized in embodiment 2 is related to, to dog bocavirus,
Canine parvovirus, canine distemper virus, canine infectious hepatitis virus, canine parainfluenza virus, feline distemper virus etc. carry out PCR amplification,
To detect the specificity of the kit.The result is shown in Figure 1.
Fig. 1 gel electrophoresis result shows that only expected size (400bp or so) occurs in dog bocavirus positive recombinant plasmid
Amplification purpose band, and canine parvovirus, canine distemper virus, canine infectious hepatitis virus, canine parainfluenza virus, cat distemper pyreticosis
The not shown band such as poison shows that the kit has preferable specificity.
4 sensitivity tests of embodiment
Choosing dog bocavirus positive recombinant plasmid is template (concentration is 480.62ng/ μ L), carries out doubling dilution (respectively
It is 100Again, 101Again, 102Again, 103Again, 104Again, 105Again, 106Again, 107Again, 108Again, 109Again, 1010Dilute again), to measure
The sensibility of this method.As a result see Fig. 2.
Fig. 2 is as the result is shown: the result of swimming lane 6 is highest extension rate 105, that is, optimize after kit sensibility be
4.8062pg/μL。
The detection of 5 clinical sample of embodiment
Clinical 425 parts of fecal sample of dog is acquired from Guangzhou surrounding area, carries out PCR detection.
Through detecting, there is 1 part of dog bocavirus positive sample in 425 parts of clinical samples, further through nucleic acid sequencing, determining should
Sample infects 2 type of dog bocavirus (CBoV2).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
<120>universal PCR detection primer, kit and the detection method of a kind of dog bocavirus
<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. a kind of universal PCR detection primer of dog bocavirus, nucleotide sequence are 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: the kit contains claim 1 institute
The primer stated.
3. a kind of universal PCR detection method of dog bocavirus, characterized in that it comprises the following steps:
1) viral nucleic acid is extracted from sample；
2) using nucleic acid as template, pcr amplification reaction is carried out with primer pair CBoV-F and CBoV-R described in claim 1 and is expanded
Increase production object；
3) pcr amplification product is subjected to agarose gel electrophoresis analysis, the observation under gel imaging system is as a result, determine virus type
Type；
The above method is not suitable for disease diagnosis and therapy.
4. PCR detection method according to claim 3, which is characterized in that pcr amplification reaction system in step 2) are as follows:
2 × Taq PCR Master Mix 12.5 μ L, ddH28.5 μ l of O, DNA profiling 0.5 μ L of 3 μ L, primer CBoV-F, primer
0.5 μ L of CBoV-R, total volume are 25 μ L.
5. PCR detection method according to claim 3, which is characterized in that PCR response procedures in step 2): 95 DEG C of pre- changes
Property 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s amount to 40 circulations；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 |
|---|
| Epidemiological investigation reveals genetic diversity and high co-infection rate of canine bocavirus strains circulating in Heilongjiang province, Northeast China;Donghua Guo et al;《Research in Veterinary Science》;20160630;第106卷;第7-13页 * |
| First complete genome sequence of canine bocavirus 2 in mainland China;S.-L.Zhai et al;《New Microbe and New Infect》;20170413;第18卷;第47-49页 * |
| 犬博卡病毒PCR检测方法的建立及其应用;陈意伟等;《安徽农业科学》;20160430;第44卷(第10期);第148-149页 * |
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