CN106148332A - A kind of Cyprinus carpio herpes virus type 2 CPA detection primer and application - Google Patents
A kind of Cyprinus carpio herpes virus type 2 CPA detection primer and application Download PDFInfo
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Abstract
The invention discloses CPA detection primer and the application of a kind of Cyprinus carpio herpes virus type 2.A kind of Cyprinus carpio herpes virus type 2 CPA detection kit, including following component: 10 × ThermoPol®Reaction Buffer；Bst archaeal dna polymerase；dNTPs；Cross primer 1S；Probe primer 2A, 3A；Peel off primer 4S, 5A；MgSO4；Betaine；Nucleic acid detection test strip.The present invention has simplicity, quick, high specific and the feature of susceptiveness, and result judges objective, directly perceived, and low cost, easy to use, the safest to human and environment.The present invention is possible not only to use in specialized laboratory, it is also possible to is applied to the field quick detection in field, it is only necessary to a metal bath or water-bath, can accurately detect the Cyprinus carpio herpes virus type 2 in sample in 1.5 hours.
Description
Technical field
The invention belongs to fishes virus detection technique field, be specifically related to a kind of Cyprinus carpio herpes virus type 2 CPA detection primer, also
Relate to the application of this primer.
Background technology
Cyprinus carpio herpes virus type 2 (CyHV-2) is the sick (herpe of simplex keratitis Hematopoietic Necrosis causing Carassius auratus mortality
Sviral haematopoietic necrosis, HVHN) cause of disease, therefore also referred to as Carassius auratus hematopoietic necrosis virus (goldfish
Haematopoietic necrosis virus, GFHNV).According to the systematic naming method rule of international virus system classification committee,
It is officially named Cyprinus carpio herpes virus type 2 (Cyprinid herpesvirus 2, CyHV-2).This virus is autumn in 1992 and 1
993 year spring was found first because of the Carassius auratus mortality that causes western Japan to cultivate, and its fatality rate is up to 100%.This disease
Subsequently in the U.S., TaiWan, China, Australia and Britain are broken out in succession, cause huge economic loss to Carassius auratus cultivation.Closely
Nian Lai, the Carassius auratus of the main culture zone of China Carassius auratus (Carassius aurutus gibelio) occurs in that massive mortality, mortality rate reach more than 80%,
Through electron microscopic observation and Molecular Detection, determine that cause of disease is CyHV-2.This disease spread scope is wide, infectiousness is strong, rapid onset, cause
Dead rate is high, causes huge economic loss to China's crucian cultivation industry, become China's aquaculture endangers the most serious
One of viral disease.
Cell culture of isolated technology is the effective ways of viral diagnosis, it is common that the fish that OIE (OIE) is recommended
The prefered method of viroid detection.But existing research data shows, CyHV-2 is difficult in existing fish cell system propagation.
Current existing common freshwater fish cell line, such as fat head Cyprinus carpio cell (fathead minnow cells, FHM), Cyprinus carpio epithelium
Oncocyte (epithelioma papillosum cyprini, EPC), Anguillar japonica nephrocyte (eel kidney, EK-1), salmon embryo
Fetus cells (chinook salmon embryo, CHSE-214), rainbow trout gonadal cell (rainbow trout gonad, R
And tilapia gonad cell (tilapia ovary, TO-2) etc. is all insensitive to CyHV-2 TG-2).The visible current stage,
Cell cultivation is not the effective ways of CyHV-2 diagnosis, and it is most effective that technology based on viral gene detection just becomes CyHV-2
Detection method.
Common gene amplification includes polymerase chain reaction (Polymerase Chain Reaction, PCR) and derives
Technology such as nest-type PRC, real-time fluorescence quantitative PCR etc..This kind of technology depends on the experimental apparatus such as PCR instrument of costliness with in real time
Quantitative real time PCR Instrument, although testing result is accurately and reliably, but is only suitable for the Pathogen test that Specialty Experiment is indoor.Disobey at present
The technique of gene detection in instrument and equipment, applicable on-the-spot quick diagnosis is relied to include loop-mediated isothermal amplification technology (loop-mediate
D isothermal amplification, LAMP) and cross primer amplification technique (Cross-Priming Amplification,
CPA).The feature of LAMP method is for four primers of six region designs on target gene, utilizes strand displacement type DNA to be polymerized
Enzyme carries out amplified reaction under constant temperature, and the response time is usually 1 hour, and product typically directly detects by an unaided eye white
Detect by an unaided eye again after precipitating or dyeing with Green fluorescent dye.CPA technology is that the most emerging a kind of gene isothermal expands
Increasing technology, is to be invented (invention patent mandate number: ZL 200810134583.1) by Hangzhou You Sida company, and it is for gene
Target sequence design cross primer and stripping primer, also utilize strand displacement type archaeal dna polymerase to carry out amplification under constant temperature anti-
Should, the response time is usually 1 hour.This technology introduces a pair probe primer, uses immune-gold labeled test strips to enter product
Row detection.This technology is the same with LAMP technology, eliminates the dependence to expensive instrument, but compared with LAMP technology, its
The observation of testing result is the most directly perceived, objective, convenient, only needs the presence or absence according to detecting line in test strips can clearly judge negative and positive
Property result, it is to avoid LAMP technology makes detect by an unaided eye precipitation or the subjectivity of color change.This technology is as a kind of basis
Method, when being applied to specifically detect object, is required for detecting the feature of subject gene structure, selects target gene pointedly
And amplification site, appropriate design cross primer, detection primer and peel off primer to improve the specificity of method and sensitivity, and
Optimize the concentration of other materials in each primer concentration ratio and reaction system, it is thus achieved that optimum reaction condition, and then could obtain relatively
Good Detection results.This technology is applied in the detection of CyHV-2 by the present invention, carries out method according to the feature of CyHV-2
Optimize, establish the CPA detection method of CyHV-2.This method is by design of primers link and to reaction system part
Material concentration is optimized, the accuracy of specificity and testing result to improve method.The present invention have the highest specificity and
Sensitivity, quick, easy, result judges directly perceived, objective, accurately and reliably, be suitable for Cyprinus carpio herpes virus type 2 sick on-the-spot quickly
Detection.
Summary of the invention
Object of the present invention is to provide a kind of Cyprinus carpio herpes virus type 2 (CyHV-2) CPA detection primer, according to GenBank
Helicase gene coded sequence (KC245087.1) the design CPA primer of the CyHV-2 strain announced, simultaneously to part
Base is changed avoiding mispairing and then the specificity of raising reaction between primer, utilizes the specific of CPA technology amplification target gene
Region, is used for quickly detecting Cyprinus carpio herpes virus type 2 from molecular level, has simplicity, quick, high specific and susceptiveness
Feature.
Further object is that and provide a kind of Cyprinus carpio herpes virus type 2 CPA detection primer at preparation Cyprinus carpio herpesvirus
Application in 2 type CPA detection kit.Utilize the test kit that the present invention provides, it is only necessary to a metal bath or water-bath
In 1.5 hours, accurately detect the CyHV-2 in sample, the sick fish tissues that CyHV-2 infects can be detected, CYH can be detected
The cell culture (such as GiCB cell) that V-2 infects, is highly suitable for the field quick detection of CyHV-2.
In order to achieve the above object, the present invention is by the following technical solutions:
A kind of CPA detection primer of Cyprinus carpio herpes virus type 2:
1S:5’-GTCCCTGGCAGAAATAAGCTGCAGCCATAGGACCTTCCACAT-3’
2A:5’-(Biotin)GTCCCTGGCAGAAATAAG-3’
3A:5’-(FITC)TGCCGTGCTCCAGTTTCA-3’
4S:5’-ATGTGTTGTGTTGTGTTTGTG-3’
5A:5’-ACTCGGTTTGATGGGACT-3’
The application in preparation Cyprinus carpio herpes virus type 2 CPA detection kit of a kind of Cyprinus carpio herpes virus type 2 CPA detection primer,
Described test kit includes:Reaction Buffer；Bst archaeal dna polymerase；dNTPs；Intersection is drawn
Thing 1S；Probe primer 2A, 3A；Peel off primer 4S, 5A；MgSO4；Betaine；Nucleic acid detection test strip.
1S:5’-GTCCCTGGCAGAAATAAGCTGCAGCCATAGGACCTTCCACAT-3’
2A:5’-(Biotin)GTCCCTGGCAGAAATAAG-3’
3A:5’-(FITC)TGCCGTGCTCCAGTTTCA-3’
4S:5’-ATGTGTTGTGTTGTGTTTGTG-3’
5A:5’-ACTCGGTTTGATGGGACT-3’
Utilize Cyprinus carpio herpes virus type 2 CPA detection primer or test kit nondiagnostic detection Cyprinus carpio herpes virus type 2 method:
1, the acquisition of viral DNA:
2, CPA amplification:
Use 25 μ L reaction systems, including:Reaction Buffer 2.5 μ L, cross primer 1S 1.0
μM, each 0.2～1.0 μM of probe primer 2A, 3A, peels off each 0.2～1.0 μM of primer 4S, 5A, MgSO44～12mM,
DNTPs 0.2～1.0mM, Betaine 0.7M, Bst archaeal dna polymerase 8U, template DNA 1 μ L, ddH2O complements to 2
5μL；The temperature range of 55～65 DEG C, after reacting 30～90 minutes, 80 DEG C inactivate 2 minutes.
3, testing result judges, can carry out the judgement of result by one of following two kinds of methods:
1) take amplified production, after the agarose gel electrophoresis with 2.0% (W/V), be placed in imaging in gel imaging system, electricity
Swimming picture display CPA characteristic scalariform band, then result is positive；Without any band, then result is negative.
2) take amplified production, detect by nucleic acid detection test strip, only in quality control region C, one red line occurs, represent sample detection
Result is negative.Two red lines, a detection line, a nature controlling line occur, represents that sample detection result is positive.In quality control region C
Redfree lines occur, showing that nucleic acid detection test strip lost efficacy, testing result is invalid.
Procedure described above, in the CPA amplification of step 2, its system is preferably:
Use 25 μ L reaction systems, including:Reaction Buffer 2.5 μ L, cross primer 1S 1.0
μM, each 0.4 μM of probe primer 2A, 3A, peels off each 0.4 μM of primer 4S, 5A, MgSO46.0mM, dNTPs 0.
8mM, Betaine 0.7M, Bst archaeal dna polymerase 8U, template DNA 1 μ L, ddH2O complements to 25 μ L；63 DEG C anti-
After answering 60 minutes, 80 DEG C inactivate 2 minutes.
Compared with prior art, the invention have the advantages that
1, specificity is good, can effectively detect Cyprinus carpio herpes virus type 2；
2, rapidly and efficiently, detect about 1.5 hours time, be highly suitable for the field quick detection of CyHV-2；
3, being independent of instrument and equipment and the specialty testing staff of costliness, testing cost is low；
4, testing result judges simple, objective, directly perceived.
The most highly sensitive, the CyHV-2 to 10copies/ μ L can be detected.
Detailed description of the invention
Following Examples further illustrates the present invention, but should not regard limitation of the present invention.Technical scheme of the present invention, as
Not specified, it is the conventional scheme of this area；Described reagent or material, if not otherwise specified, the most disclosed or derive from
Commercial channel.
Embodiment 1:
A kind of Cyprinus carpio herpes virus type 2 CPA detection kit, including:
Reaction Buffer；Bst archaeal dna polymerase (NEB)；dNTPs(Takara)；Intersection is drawn
Thing 1S；Probe primer 2A, 3A；Peel off primer 4S, 5A；MgSO4(Promega)；Betaine(Sigma)；Nucleic acid is examined
Test paper slip (Hangzhou You Sida company).
1S:5’-GTCCCTGGCAGAAATAAGCTGCAGCCATAGGACCTTCCACAT-3’
2A:5’-(Biotin)GTCCCTGGCAGAAATAAG-3’
3A:5’-(FITC)TGCCGTGCTCCAGTTTCA-3’
4S:5’-ATGTGTTGTGTTGTGTTTGTG-3’
5A:5’-ACTCGGTTTGATGGGACT-3’
Embodiment 2:
Different primers concentration and the optimization of ratio in a kind of Cyprinus carpio herpes virus type 2 CPA detection kit:
One, take measuring samples extract viral DNA:
Take Cyprinus carpio herpes virus type 2 (CyHV-2) (creep, Zeng Lingbing, Yang Deguo, etc. Cyprinus carpio herpes virus type 2 Wuhan strain point
From with identify [J]. Chinese aquatic science, 2013,20 (6): 1303-1309) tissue such as the Carassius auratus disease fish gill that infects, spleen, kidney is even
Serosity 300 μ L, usesReagent or Viral DNA Kit test kit extract DNA to specifications, are finally dissolved in 30
μ L aquesterilisa ,-20 DEG C save backup.
Two, the reaction system of CPA amplification:
Use 25 μ L reaction systems, PCR pipe add viral DNA template 1 μ L,Reaction
Buffer 2.5 μ L, cross primer 1S, probe primer 2A, 3A, peel off primer 4S, 5A, MgSO46.0mM, dNTP
S 0.8mM, Betaine 0.7M, Bst archaeal dna polymerase 8U, nuclease free water complements to 25 μ L.To arrange blank right simultaneously
According to.
Wherein cross primer (1S), probe primer (2A, 3A), peel off primer (4S, 5A) and use different concentration ratio:
1) 1S:1.0 μM, 2A/3A:0.2 μM, 4S/5A:0.2 μM
2) 1S:1.0 μM, 2A/3A:0.4 μM, 4S/5A:0.4 μM
3) 1S:1.0 μM, 2A/3A:0.6 μM, 4S/5A:0.6 μM
4) 1S:1.0 μM, 2A/3A:0.8 μM, 4S/5A:0.8 μM
5) 1S:1.0 μM, 2A/3A:1.0 μM, 4S/5A:1.0 μM
Three, the reaction condition of CPA amplification:
Reaction tube is in 63 DEG C of incubation 60min, 80 DEG C of inactivation 2min.
Four, testing result judges:
Take 5 μ L amplified productions, after the agarose gel electrophoresis of 2.0%, be placed in imaging in gel imaging system, electrophoresis picture
It is best that display primer concentration combines 2 expanding effects, it may be assumed that 1S:1.0 μM, 2A/3A:0.4 μM, 4S/5A:0.4 μM.
Embodiment 3:
The MgSO of variable concentrations in a kind of Cyprinus carpio herpes virus type 2 CPA detection kit4Optimization:
One, take measuring samples extract viral DNA:
Preparation method is with embodiment 2.
Two, the reaction system of CPA amplification:
Use 25 μ L reaction systems, PCR pipe add viral DNA template 1 μ L,Reaction
Buffer 2.5 μ L, 1S 1.0 μMs, 2A/3A 0.4 μM, 4S/5A 0.4 μM, MgSO4, dNTPs 0.8mM, Betain
E 0.7M, Bst archaeal dna polymerase 8U, nuclease free water complements to 25 μ L.Blank is set simultaneously.
Wherein MgSO4Concentration be respectively 4.0,6.0,8.0,10.0,12.0mM.
Three, the reaction condition of CPA amplification:
Reaction tube is in 63 DEG C of incubation 60min, 80 DEG C of inactivation 2min.
Four, testing result judges:
Take 5 μ L amplified productions, after the agarose gel electrophoresis of 2.0%, be placed in imaging in gel imaging system, electrophoresis picture
Display MgSO4Concentration when being 6.0mM effect best.
Embodiment 4:
The optimization of variable concentrations dNTPs in a kind of Cyprinus carpio herpes virus type 2 CPA detection kit:
One, take measuring samples extract viral DNA:
Preparation method is with embodiment 2.
Two, the reaction system of CPA amplification:
Use 25 μ L reaction systems, PCR pipe add viral DNA template 1 μ L,Reaction B
Uffer 2.5 μ L, 1S 1.0 μMs, 2A/3A 0.4 μM, 4S/5A 0.4 μM, MgSO46.0mM, dNTPs, Betaine 0.
7M, Bst archaeal dna polymerase 5.6U, nuclease free water complements to 25 μ L.Blank is set simultaneously.
Wherein the concentration of dNTPs be respectively 0.2,0.4,0.6,0.8,1.0mM.
Three, the reaction condition of CPA amplification:
Reaction tube is in 63 DEG C of incubation 60min, 80 DEG C of inactivation 2min.
Four, testing result judges:
Take 5 μ L amplified productions, after the agarose gel electrophoresis of 2.0%, be placed in imaging in gel imaging system, electrophoresis picture
When display dNTPs concentration is 0.8mM, effect is best.
Embodiment 5:
The optimization of differential responses temperature in a kind of Cyprinus carpio herpes virus type 2 CPA detection kit:
One, take measuring samples extract viral DNA:
Preparation method is with embodiment 2.
Two, the reaction system of CPA amplification:
Use 25 μ L reaction systems, PCR pipe add viral DNA template 1 μ L,Reaction B
Uffer 2.5 μ L, 1S 1.0 μMs, 2A/3A 0.4 μM, 4S/5A 0.4 μM, MgSO46.0mM, dNTPs 0.8mM, B
Etaine 0.7M, Bst archaeal dna polymerase 8U, nuclease free water complements to 25 μ L.Blank is set simultaneously.
Three, the reaction condition of CPA amplification:
Reaction tube be respectively placed in 55 DEG C, 57 DEG C, 59 DEG C, 61 DEG C, 63 DEG C, after 65 DEG C of incubation 60min, 80 DEG C of inactivation 2min.
Four, testing result judges:
Take 5 μ L amplified productions, after the agarose gel electrophoresis of 2.0%, be placed in imaging in gel imaging system, electrophoresis picture
When display reaction temperature is 63 DEG C, effect is best.
Embodiment 6:
A kind of specificity of Cyprinus carpio herpes virus type 2 CPA detection kit:
1, take measuring samples and extract viral DNA or bacterial nucleic acid:
CyHV-2 (creep, Zeng Lingbing, Yang Deguo, etc. the separation of Cyprinus carpio herpes virus type 2 Wuhan strain and qualification [J]. China Water
Obstetrics, 2013,20 (6): 1303-1309) (Zhu Xia, Li Xinwei, Wang Hao, etc. a. strain for the sick fish tissues homogenate that infects, KHV
The separation of Koi herpesvirus with identify [J]. China's Preventive Veterinary Medicine report, 2011,33 (5): 340-343) the Koi-Fin cell that infects,
Eel herpesvirus (HVA) (Jakob E, Neuhaus H, Steinhagen D, et al.Monitoring of Herpesvirus
anguillae(HVA)infections in European eel,Anguilla anguilla(L.),in northern Germany[J].
J Fish Dis, 2009,32 (6): 557-561) the EPC cell that infects, GSIV (China typical culture collection center, CC
TCC NO:V201134) infect EPC cell, white spot syndrome virus (WSSV) (creep, Fan Yuding, Zhou Yong,
Deng. Standard PCR and labelled by nested-PCR method quickly detect Procambrus clarkii white spot syndrome virus (WSSV) [J]. fresh water fishery,
2008,38 (6): 52-54.) the sick shrimp tissue homogenate infected, in-80 DEG C to room temperature multigelation 3 times, 5000r/min
Centrifugal 30min, takes supernatant 300 μ L, extracts DNA to specifications with Viral DNA Kit test kit, be finally dissolved in 30 μ
L aquesterilisa ,-20 DEG C save backup.
Additionally culture of isolated is from a kind of pathogenic bacterium of Andrias davidianus Blanchard: Aeromonas hydrophila (Ah) (Meng Yan, Zeng Lingbing, Yang Yanqing, etc. big
The separation of salamander Characteristics of Pathogenic Edwardsiella tarda bacterium and identification research [J]. Journal of Northwest Sci Tech University of Agriculture and Forestry (natural science edition), 2009,37 (3):
77-81.) Conventional bacteria is cultivated, directly with bacterium solution (109CFU/mL) as template detection.
2, the reaction system of CPA amplification:
Use 25 μ L reaction systems, PCR pipe add viral DNA template 1 μ L,Reaction B
Uffer 2.5 μ L, 1S 1.0 μMs, 2A/3A 0.4 μM, 4S/5A 0.4 μM, MgSO46.0mM, dNTPs 0.8mM, B
Etaine 0.7M, Bst archaeal dna polymerase 8U, nuclease free water complements to 25 μ L.Blank is set simultaneously.
3, the reaction condition of CPA amplification:
After reaction tube is respectively placed in 63 DEG C of incubation 60min, 80 DEG C of inactivation 2min.
4, testing result judges:
Detect by nucleic acid detection test strip, test strips lower end dropping 5～8 μ L amplified productions, then (the excellent think of in Hangzhou reaches with buffer
Company) 3 droplets, can with the naked eye interpretation after 3～5min.Nucleic acid detection test strip shows, for the specific C of CyHV-2 design
PA primer sets can guarantee that the specific detection to CyHV-2, only detects CyHV-2, and KHV, HVA, GSIV, W
SSV, Ah, blank (water) are feminine gender.
Embodiment 7:
A kind of Cyprinus carpio herpes virus type 2 CPA detection kit sensitivity:
1, CyHV-2Helicase gene clone and standard construction of recombinant plasmid:
Extracting CyHV-2 virus STb gene (preparation method is with embodiment 2), PCR expands Helicase gene (KC24508
7.1) full length sequence, and clone into pMD19-T plasmid, build CyHV-2Helicase gene standard recombiant plasmid.Extract
And purification of Recombinant plasmid, measure plasmid concentration, calculate plasmid copy number, and with pure water, standard plasmid concentration is adjusted to 1010c
opies/μL.During sensitivity determination, standard plasmid pure water being carried out 10 times of gradient dilutions, making plasmid concentration scope is 109co
Pies/ μ L～100Copies/ μ L, as the DNA profiling of CPA reaction.
2, the reaction system of CPA amplification:
Use 25 μ L reaction systems, PCR pipe add viral DNA template 1 μ L,Reaction B
Uffer 2.5 μ L, 1S 1.0 μMs, 2A/3A 0.4 μM, 4S/5A 0.4 μM, MgSO46.0mM, dNTPs 0.8mM, B
Etaine 0.7M, Bst archaeal dna polymerase 8U, nuclease free water complements to 25 μ L.Blank is set simultaneously.
3, the reaction condition of CPA amplification:
Reaction tube is in 63 DEG C of incubation 60min, 80 DEG C of inactivation 2min.
4, testing result judges:
Detect by nucleic acid detection test strip, test strips lower end dropping 5～8 μ L amplified productions, then with buffer 3,3～5min
After can with the naked eye interpretation.Nucleic acid detection test strip show can lowest detection to the CyHV-2 of 10copies/ μ L.
SEQUENCE LISTING
<110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120>a kind of Cyprinus carpio herpes virus type 2 CPA detection primer and application
<130>a kind of Cyprinus carpio herpes virus type 2 CPA detection primer and application
<160> 5
<170> PatentIn version 3.1
<210> 1
<211> 42
<212> DNA
<213>synthetic
<400> 1
gtccctggca
gaaataagct gcagccatag gaccttccac at 42
<210> 2
<211> 18
<212> DNA
<213>synthetic
<400> 2
gtccctggca
gaaataag
18
<210> 3
<211> 18
<212> DNA
<213>synthetic
<400> 3
tgccgtgctc
cagtttca
18
<210> 4
<211> 21
<212> DNA
<213>synthetic
<400> 4
atgtgttgtg
ttgtgtttgt g
21
<210> 5
<211> 18
<212> DNA
<213>synthetic
<400> 5
actcggtttg
atgggact
18
Claims (5)
1. the CPA detection primer of a Cyprinus carpio herpes virus type 2:
1S: 5’-GTCCCTGGCAGAAATAAGCTGCAGCCATAGGACCTTCCACAT-3’
2A: 5’-(Biotin) GTCCCTGGCAGAAATAAG-3’
3A: 5’-(FITC) TGCCGTGCTCCAGTTTCA-3’
4S: 5’-ATGTGTTGTGTTGTGTTTGTG-3’
5A: 5’-ACTCGGTTTGATGGGACT-3’。
2. the application in preparation Cyprinus carpio herpes virus type 2 CPA detection kit of the Cyprinus carpio herpes virus type 2 CPA detection primer described in claim 1.
Application the most according to claim 2, described Cyprinus carpio herpes virus type 2 CPA detection kit, including:
10×ThermoPol®Reaction Buffer；Bst archaeal dna polymerase；dNTPs；Cross primer 1S；Probe primer 2A, 3A；Peel off primer 4S, 5A；MgSO4；Betaine；Nucleic acid detection test strip；
1S: 5’-GTCCCTGGCAGAAATAAGCTGCAGCCATAGGACCTTCCACAT-3’
2A: 5’-(Biotin) GTCCCTGGCAGAAATAAG-3’
3A: 5’-(FITC) TGCCGTGCTCCAGTTTCA-3’
4S: 5’-ATGTGTTGTGTTGTGTTTGTG-3’
5A: 5’-ACTCGGTTTGATGGGACT-3’。
4. the method utilizing the detection Cyprinus carpio herpes virus type 2 of primer nondiagnostic described in claim 1:
1, the acquisition of viral DNA:
2, CPA amplification:
Use 25 μ L reaction systems, including: 10 × ThermoPol®
Reaction Buffer 2.5 μ L, cross primer 1S 1.0 μMs, each 0.2 ~ 1.0 μM of probe primer 2A, 3A, peel off each 0.2 ~ 1.0 μM of primer 4S, 5A, MgSO44 ~ 12mM, dNTPs 0.2 ~ 1.0mM, Betaine 0.7M, Bst archaeal dna polymerase 8U, template DNA 1 μ L, ddH2O complements to 25 μ L；The temperature range of 55 ~ 65 DEG C, after reacting 30 ~ 90 minutes, 80 DEG C inactivate 2 minutes；
3, testing result judges, can carry out the judgement of result by one of following two kinds of methods:
1) taking amplified production, after the agarose gel electrophoresis of 2.0%, be placed in imaging in gel imaging system, electrophoresis picture display CPA characteristic scalariform band, then result is positive；Without any band, then result is negative；
2) take amplified production, detect by nucleic acid detection test strip, only in quality control region C, one red line occurs, represent that sample detection result is negative；Two red lines, a detection line, a nature controlling line occur, represents that sample detection result is positive；Occurring at quality control region C redfree lines, showing that nucleic acid detection test strip lost efficacy, testing result is invalid.
Method the most according to claim 4, step 2 CPA amplification reaction system and reaction condition be:
Use 25 μ L reaction systems, including: 10 × ThermoPol®
Reaction Buffer 2.5 μ L, cross primer 1S 1.0 μMs, each 0.4 μM of probe primer 2A, 3A, peel off each 0.4 μM of primer 4S, 5A, MgSO46.0mM, dNTPs 1.0mM, Betaine 0.7M, Bst archaeal dna polymerase 8U, template DNA 1 μ L, ddH2O complements to 25 μ L；After 63 DEG C are reacted 60 minutes, 80 DEG C inactivate 2 minutes.
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| CN107419006A (en) * | 2017-05-19 | 2017-12-01 | 中山大学 | The CPA primer sets and its detection method of a kind of quick detection legionella pneumophilia |
| CN108103246A (en) * | 2018-02-01 | 2018-06-01 | 上海海洋大学 | For detecting the RPA kits of II type carp herpesvirals and its primer special and probe |
| CN109666749A (en) * | 2018-09-29 | 2019-04-23 | 四川大学华西医院 | A kind of kit and method detecting Acinetobacter bauamnnii |
| CN110894554A (en) * | 2019-11-18 | 2020-03-20 | 浙江省淡水水产研究所 | Primer probe set for detecting carp herpesvirus II, kit and application |
| CN111635961A (en) * | 2020-06-30 | 2020-09-08 | 广东省农业科学院动物卫生研究所 | Koi herpesvirus detection kit and detection method |
| CN112725530A (en) * | 2021-01-19 | 2021-04-30 | 中国计量大学 | Cross primer amplification primer group for detecting bean pod mottle virus, kit and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638685A (en) * | 2008-07-29 | 2010-02-03 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
-
2015
- 2015-04-23 CN CN201510196679.0A patent/CN106148332B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638685A (en) * | 2008-07-29 | 2010-02-03 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG,H 等: "GenBank 登录号：KC245087.1", 《NCBI》 * |
| 徐进 等: "鲤疱疹病毒2型武汉株的分离与鉴定", 《中国水产科学》 * |
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| CN106755591A (en) * | 2017-02-06 | 2017-05-31 | 广东省农业科学院动物卫生研究所 | Detect intersection isothermal duplication primer sets, kit and the application of Pseudorabies virus street strain |
| CN107419006A (en) * | 2017-05-19 | 2017-12-01 | 中山大学 | The CPA primer sets and its detection method of a kind of quick detection legionella pneumophilia |
| CN108103246A (en) * | 2018-02-01 | 2018-06-01 | 上海海洋大学 | For detecting the RPA kits of II type carp herpesvirals and its primer special and probe |
| CN109666749A (en) * | 2018-09-29 | 2019-04-23 | 四川大学华西医院 | A kind of kit and method detecting Acinetobacter bauamnnii |
| CN110894554A (en) * | 2019-11-18 | 2020-03-20 | 浙江省淡水水产研究所 | Primer probe set for detecting carp herpesvirus II, kit and application |
| CN110894554B (en) * | 2019-11-18 | 2023-05-02 | 浙江省淡水水产研究所 | Primer probe group for detecting cyprinid herpesvirus II, kit and application |
| CN111635961A (en) * | 2020-06-30 | 2020-09-08 | 广东省农业科学院动物卫生研究所 | Koi herpesvirus detection kit and detection method |
| CN111635961B (en) * | 2020-06-30 | 2024-05-10 | 广东省农业科学院动物卫生研究所 | Kit and method for detecting koi herpesvirus |
| CN112725530A (en) * | 2021-01-19 | 2021-04-30 | 中国计量大学 | Cross primer amplification primer group for detecting bean pod mottle virus, kit and application |
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