CN107746898A - A kind of detection kit and detection method for detecting carp edema virus - Google Patents
A kind of detection kit and detection method for detecting carp edema virus Download PDFInfo
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- CN107746898A CN107746898A CN201711075343.4A CN201711075343A CN107746898A CN 107746898 A CN107746898 A CN 107746898A CN 201711075343 A CN201711075343 A CN 201711075343A CN 107746898 A CN107746898 A CN 107746898A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of detection kit and detection method for detecting carp edema virus, the kit includes the first inner primer and the second inner primer respectively containing sequence shown in 528 FIP, 528 BIP, 528 F3,528 B3 and 528 LF, the first outer primer and the second outer primer and ring primer；Wherein：Sequence shown in 528 F3 is：TCCCACAGAATGTAATCTCAA；Sequence shown in 528 FIP is：GTCTGTCTTCGATAAGACAATGACT‑TGTGGAGTTTTTGAAGTATACTGT；Sequence shown in 528 LF is：TGCTCTAGTTCTAGGATTGT；Sequence shown in 528 BIP is：TCAATCTGAATTCCTTTCCAGAACA‑CGTTGATACAATTCCAGAGC；Sequence shown in 528 B3 is：ACCTCATCCAAATTACGTAGT.
Description
Technical field
The present invention relates to virus detection techniques field, and in particular to a kind of detection kit for detecting carp edema virus and
Detection method.
Background technology
Carp edema virus (Carp Edema Virus, CEV) infection can cause carp edema disease (Koi sleepy
Disease, KSD), it is to endanger carp, a kind of virosis of fancy carp, is found in Hiroshima,Japan and Nigata earliest within 1972, and
Japanese whole sprawling, causes carp and the fancy carp mortality of cultivation.In recent years on Germany, France, Britain, Holland, Czech, ground difficult to understand
The states such as profit, Turkey find that the carp cultivated and fancy carp cause fulminant dead because of the disease.
Epidemic disease is broken out in July, 2014 plant of Fangshan District of Beijing discovery Crucian carp fingerlings in China, more than 50% in 3 days
Juvenile fish it is dead, symptom infects similar to Koi herpesvirus (Koi Herpes Virus, KHV), but is determined not through PCR detections
It is KHVD.After further research, it is observed that poxvirus sample particle from internal organ section, with PCR and Real-time PCR sides
Method can detect the genetic fragment of carp edema virus, and the respective segments coincidence rate of known carp edema viral (CEV)
100%, by electron microscopic observation, PCR amplifications, sequencing and comparison, it is by carp edema virus to determine Fangshan morbidity fancy carp
(CEV) carp edema disease caused by infection.
Carp edema virus is a kind of poxvirus, and poxvirus is a kind of important virus for seriously endangering animal, but due to
Think that carp edema disease caused by the virus infection of carp edema is limited only to Japanese native country always before, and carp edema disease
Illness and Koi herpesvirus sick (Koi Herpes Virus Disease, KHVD) are very similar, clinically easily obscure, give
Detection and prevention and control add difficulty.
Detect in the prior art the common laboratory detection method of carp edema virus include electron microscopic observation, cell culture,
Rely primarily on histopathology, electron microscopic observation, regular-PCR, sleeve type PCR and fluorescence quantitative PCR detection technique.But such side
There is limitation in method, or time-consuming longer or detection sensitivity is relatively low or requires professional and technical personnel or needs in clinical detection
Expensive professional auxiliary equipment is wanted, is not suitable for the quick diagnosis examination CEV at scene.
Cell be separately cultured be currently fishes virus detection technical way, and be acknowledged as " goldstandard ", but real
The CEV for trampling discovery does not occur CPE in EPC, FHM, CAR cell, prompts CEV may be insensitive to above-mentioned cell.To one
A bit can not in cell culture of isolated infection aquatic animal virus, by typical clinical symptom combination PCR identify can be right
Case is made a definite diagnosis, and to no clinical symptoms, can identify and be sequenced to make a definite diagnosis by PCR, and detection, identification for CEV can
The method that clinical symptoms according to morbidity fish expand CEV specific bands by PCR simultaneously.
Fancy carp and carp are the important breed varieties of China, do not have active drug control after being broken out due to viral disease, because
Carp edema disease caused by this fancy carp and the virosis of carp particularly carp edema viral (CEV), the spy to be gone together both at home and abroad
Do not pay close attention to.Due to not effective treatment method, can only aim at prevention, thus the diagnosis of virus turns into detection technique research
Very active field in fishes virus research.Carp edema virus how is rapidly detected, is this area urgent need to resolve
Technical problem.
Ring mediation isothermal amplification (LAMP) be by T.Notomi (Notomi T, Okayama H,
MasubuchiH, et al.Loop-mediated isothermal amplification of DNA.Nucleic Acids
Res 2000；28(12)：63) a kind of novel nucleic acids amplification technique of invention, the technology rely on 4 primers specifically designed and one
Kind of the archaeal dna polymerase with strand displacement characteristic, under isothermal conditions can efficiently, quick, height specifically expand target sequence.At present
The technology is applied to the aquatic products such as baculovirus of prawn, SVCV, Koi herpesvirus disease, giant salamander frog virus
The diagnosis of pathogen.But so far, it yet there are no and asked for detecting the LAMP primer specials of carp edema virus with kit
Generation.
The content of the invention
In order to overcome problems of the prior art, the present invention provides a kind of detection reagent for detecting carp edema virus
Box and detection method, the detection kit and detection method of detection carp edema virus being capable of quick detection carp edema diseases
Poison, it is significant to Outbreak investigation, field diagnostic, epidemiological study and disease surveillance.
To achieve the above object, the detection kit of detection carp edema virus of the present invention includes containing respectively
The first inner primer and the second inner primer of sequence shown in 528-FIP, 528-BIP, 528-F3,528-B3 and 528-LF, outside first
Primer and the second outer primer and ring primer；Wherein：
Sequence is shown in 528-F3：TCCCACAGAATGTAATCTCAA
Sequence is shown in 528-FIP：
GTCTGTCTTCGATAAGACAATGACT-TGTGGAGTTTTTGAAGTATACTGT
Sequence is shown in 528-LF：TGCTCTAGTTCTAGGATTGT
Sequence is shown in 528-BIP：
TCAATCTGAATTCCTTTCCAGAACA-CGTTGATACAATTCCAGAGC
Sequence is shown in 528-B3：ACCTCATCCAAATTACGTAGT.
The detection kit of described detection carp edema virus is reacted for ring mediated constant temperature nucleic acid amplification, and 528-
Sequence shown in FIP is the first inner primer, and sequence shown in 528-BIP is the second inner primer, and sequence shown in 528-F3 is to draw outside first
Thing, sequence shown in 528-B3 are the second outer primer, and sequence shown in 528-LF is ring primer.
P4a (528bp) the gene piece of the detection kit of described detection carp edema virus for carp edema virus
Section design primer, target sequence are as follows：
ATGGAGTATCCAAAGTACTTAGATTAATGTTATCAATGAAATTTGTGTATTGTGTTTTTGTTAGTCCAAGAGTTTTC
TTCTCATCGTTTGTTACTTTTTGTAGTTGCTTAATATTTGTGATAAGATTTCCATTGGCATAAAATCCTTCCCAAAT
TTGTGTTGAAACATGTTTTAGTGTTTTGTAGATTGTAGCATTTCCTAGTTTGTATGGCAAGAAACAAACTCTCTTTA
CTGAAACTCCTTGAGGAATTTGATCTAGAATCCCACAGAATGTAATCTCAAATTTGTTTGTGGAGTTTTTGAAGTAT
ACTGTTTCATCATACAATCCTAGAACTAGAGCAAGATTAGAAGTCATTGTCTTATCGAAGACAGACATCTTATTCCA
ATCATCAATCTGAATTCCTTTCCAGAACATAACATTTGCAATTTTAACTTGCTCTGGAATTGTATCAACGTATCCAA
TATCTTTCTTTACTACGTAATTTGGATGAGGTAGTACTTTGCTAACAAAGTCACAATAGTGAAGAG。
Sequence to be amplified is divided into seven independent regions, root by the detection kit of described detection carp edema virus
Separately design 5 primers needed for reaction according to this seven regions, including the first inner primer and the second inner primer, the first outer primer and
Second outer primer and ring primer.
The detection kit of described detection carp edema virus generates Mg during the course of the reaction2P2O7I.e. magnesium pyrophosphate is white
Color is precipitated, and the yin and yang attribute of reaction is judged by turbidity change.
The present invention also provides a kind of detection method for detecting carp edema virus, passes through the P4a to carp edema virus
(528) genetic fragment design primer, target sequence are as follows：
ATGGAGTATCCAAAGTACTTAGATTAATGTTATCAATGAAATTTGTGTATTGTGTTTTTGTTAGTCCAAGAGTTTTC
TTCTCATCGTTTGTTACTTTTTGTAGTTGCTTAATATTTGTGATAAGATTTCCATTGGCATAAAATCCTTCCCAAAT
TTGTGTTGAAACATGTTTTAGTGTTTTGTAGATTGTAGCATTTCCTAGTTTGTATGGCAAGAAACAAACTCTCTTTA
CTGAAACTCCTTGAGGAATTTGATCTAGAATCCCACAGAATGTAATCTCAAATTTGTTTGTGGAGTTTTTGAAGTAT
ACTGTTTCATCATACAATCCTAGAACTAGAGCAAGATTAGAAGTCATTGTCTTATCGAAGACAGACATCTTATTCCA
ATCATCAATCTGAATTCCTTTCCAGAACATAACATTTGCAATTTTAACTTGCTCTGGAATTGTATCAACGTATCCAA
TATCTTTCTTTACTACGTAATTTGGATGAGGTAGTACTTTGCTAACAAAGTCACAATAGTGAAGAG；
Sequence to be amplified is divided into seven independent regions, 5 according to needed for this seven regions separately design reaction
Primer, including the first inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer, for specific
Expand target gene；
The first described inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer specificity are known
Seven isolated areas on other target sequence, cause self-loopa strand replacement reaction in the presence of Bst Large fragment polymerases, 60~
Isothermal reaction 60min in the range of 67 DEG C, magnesium pyrophosphate while synthesizing target dna with accessory substance white, which precipitates, to be produced；
Using distilled water as negative control；
The yin and yang attribute of reaction is judged by turbidity change.
Calcein color change detection yin and yang attribute is preferably based on, calcein is a kind of amplification indicator, when negative
It is green when positive for light gray.
The first described inner primer contains the sequence shown in 528-FIP, and the second inner primer contains the sequence shown in 528-BIP
Row, the first outer primer contain the sequence shown in 528-F3, and the second outer primer contains the sequence shown in 528-B3, described ring primer
Contain the sequence shown in 528-LF；Wherein：
Sequence is shown in 528-F3：TCCCACAGAATGTAATCTCAA
Sequence is shown in 528-FIP：
GTCTGTCTTCGATAAGACAATGACT-TGTGGAGTTTTTGAAGTATACTGT
Sequence is shown in 528-LF：TGCTCTAGTTCTAGGATTGT
Sequence is shown in 528-BIP：
TCAATCTGAATTCCTTTCCAGAACA-CGTTGATACAATTCCAGAGC
Sequence is shown in 528-B3：ACCTCATCCAAATTACGTAGT.
The invention has the advantages that：
Ring mediation isothermal amplification (LAMP) be by T.Notomi (Notomi T, Okayama H,
MasubuchiH, et al.Loop-mediated isothermal amplification of DNA.Nucleic Acids
Res 2000；28(12):63) a kind of novel nucleic acids amplification technique of invention, the technology rely on 4 primers specifically designed and one
Kind of the archaeal dna polymerase with strand displacement characteristic, under isothermal conditions can efficiently, quick, height specifically expand target sequence.At present
The technology is applied to the aquatic products such as baculovirus of prawn, SVCV, Koi herpesvirus disease, giant salamander frog virus
The diagnosis of pathogen.But so far, it yet there are no and asked for detecting the LAMP primer specials of carp edema virus with kit
Generation.
It is of the present invention to detect the viral detection kit of carp edema and detection method compared with prior art, this hair
The CEV of bright foundation LAMP detection method only needs sample constant temperature being positioned over 63 DEG C of reaction 40-60min, it is not necessary to by it
His instrument, result can be judged according only to color change naked eyes.Method high specificity, detection sensitivity is high, operation letter
Just, quickly, available in the horizontal extensive examination for carrying out CEV in fishing ground and field diagnostic.
Calcein of the present invention adds reaction system, to result interpretation without opening reaction again since reaction
Pipe, it can be effectively prevented from causing to produce false positive because forming aerosol.
The detection technique of the present invention can realize nucleic acid amplification under isothermal conditions, and amplification procedure relies on identification target sequence seven
Individual isolated area, and amplification process is carried out under constant temperature, and detection time greatly shortens, and quick sensitive,
Can quick detection carp edema virus, there is important meaning to Outbreak investigation, field diagnostic, epidemiological study and disease surveillance
Justice.
Brief description of the drawings
Fig. 1 is the schematic diagram that the present invention carries out optimum temperature screening using nephelometry to primer.
Fig. 2 is the testing result schematic diagram that the present invention uses nephelometry detection sensitivity.
Fig. 3 is the present invention using indicator method detection Sample Negative, positive contrast's schematic diagram.
Fig. 4 is the testing result schematic diagram that the present invention uses indicator color method detection sensitivity.
Fig. 5 is that the present invention detects specific testing result schematic diagram using indicator method.
Fig. 6 is testing result schematic diagram of the present invention using indicator method detection sample.
Fig. 7 is the agarose gel electrophoresis figure of LAMP detection method product of the present invention.
1 is negative findings (light orange solution) in wherein Fig. 2, and 2 be positive findings (green solution).
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The detection kit of detection carp edema virus of the present invention include respectively containing 528-FIP, 528-BIP,
The first inner primer and the second inner primer, the first outer primer and the second outer primer of sequence shown in 528-F3,528-B3 and 528-LF
And ring primer；Wherein：
Sequence is shown in 528-F3：TCCCACAGAATGTAATCTCAA
Sequence is shown in 528-FIP：
GTCTGTCTTCGATAAGACAATGACT-TGTGGAGTTTTTGAAGTATACTGT
Sequence is shown in 528-LF：TGCTCTAGTTCTAGGATTGT
Sequence is shown in 528-BIP：
TCAATCTGAATTCCTTTCCAGAACA-CGTTGATACAATTCCAGAGC
Sequence is shown in 528-B3：ACCTCATCCAAATTACGTAGT.
The detection kit of described detection carp edema virus is reacted for ring mediated constant temperature nucleic acid amplification, and 528-
Sequence shown in FIP is the first inner primer, and sequence shown in 528-BIP is the second inner primer, and sequence shown in 528-F3 is to draw outside first
Thing, sequence shown in 528-B3 are the second outer primer, and sequence shown in 528-LF is ring primer.
P4a (528bp) the gene piece of the detection kit of described detection carp edema virus for carp edema virus
Section design primer, target sequence are as follows：
ATGGAGTATCCAAAGTACTTAGATTAATGTTATCAATGAAATTTGTGTATTGTGTTTTTGTTAGTCCAAGAGTTTTC
TTCTCATCGTTTGTTACTTTTTGTAGTTGCTTAATATTTGTGATAAGATTTCCATTGGCATAAAATCCTTCCCAAAT
TTGTGTTGAAACATGTTTTAGTGTTTTGTAGATTGTAGCATTTCCTAGTTTGTATGGCAAGAAACAAACTCTCTTTA
CTGAAACTCCTTGAGGAATTTGATCTAGAATCCCACAGAATGTAATCTCAAATTTGTTTGTGGAGTTTTTGAAGTAT
ACTGTTTCATCATACAATCCTAGAACTAGAGCAAGATTAGAAGTCATTGTCTTATCGAAGACAGACATCTTATTCCA
ATCATCAATCTGAATTCCTTTCCAGAACATAACATTTGCAATTTTAACTTGCTCTGGAATTGTATCAACGTATCCAA
TATCTTTCTTTACTACGTAATTTGGATGAGGTAGTACTTTGCTAACAAAGTCACAATAGTGAAGAG。
Sequence to be amplified is divided into seven independent regions, root by the detection kit of described detection carp edema virus
Separately design 5 primers needed for reaction according to this seven regions, including the first inner primer and the second inner primer, the first outer primer and
Second outer primer and ring primer.
The detection kit of described detection carp edema virus generates Mg during the course of the reaction2P2O7I.e. magnesium pyrophosphate is white
Color is precipitated, and the yin and yang attribute of reaction is judged by turbidity change.
The present invention also provides a kind of detection method for detecting carp edema virus, passes through the 528bp to carp edema virus
Genetic fragment designs primer, and target sequence is as follows：
ATGGAGTATCCAAAGTACTTAGATTAATGTTATCAATGAAATTTGTGTATTGTGTTTTTGTTAGTCCAAGAGTTTTC
TTCTCATCGTTTGTTACTTTTTGTAGTTGCTTAATATTTGTGATAAGATTTCCATTGGCATAAAATCCTTCCCAAAT
TTGTGTTGAAACATGTTTTAGTGTTTTGTAGATTGTAGCATTTCCTAGTTTGTATGGCAAGAAACAAACTCTCTTTA
CTGAAACTCCTTGAGGAATTTGATCTAGAATCCCACAGAATGTAATCTCAAATTTGTTTGTGGAGTTTTTGAAGTAT
ACTGTTTCATCATACAATCCTAGAACTAGAGCAAGATTAGAAGTCATTGTCTTATCGAAGACAGACATCTTATTCCA
ATCATCAATCTGAATTCCTTTCCAGAACATAACATTTGCAATTTTAACTTGCTCTGGAATTGTATCAACGTATCCAA
TATCTTTCTTTACTACGTAATTTGGATGAGGTAGTACTTTGCTAACAAAGTCACAATAGTGAAGAG；
Sequence to be amplified is divided into seven independent regions, 5 according to needed for this seven regions separately design reaction
Primer, including the first inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer, for specific
Expand target gene；
The first described inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer specificity are known
Seven isolated areas on other target sequence, cause self-loopa strand replacement reaction in the presence of Bst Large fragment polymerases, 60~
Isothermal reaction 60min in the range of 67 DEG C, magnesium pyrophosphate while synthesizing target dna with accessory substance white, which precipitates, to be produced；
Using distilled water as negative control；
The yin and yang attribute of reaction is judged by turbidity change.
Calcein color change detection yin and yang attribute is preferably based on, calcein is a kind of amplification indicator, when negative
It is green when positive for light orange.
Calcein amplification indicator is added in reaction solution, according to the color change judged result of reaction solution, green table
Show in testing sample carp edema virus be present, carp edema virus is not present in orange expression testing sample.
The first described inner primer contains the sequence shown in 528-FIP, and the second inner primer contains the sequence shown in 528-BIP
Row, the first outer primer contain the sequence shown in 528-F3, and the second outer primer contains the sequence shown in 528-B3, described ring primer
Contain the sequence shown in 528-LF；Wherein：
Sequence is shown in 528-F3：TCCCACAGAATGTAATCTCAA
Sequence is shown in 528-FIP：
GTCTGTCTTCGATAAGACAATGACT-TGTGGAGTTTTTGAAGTATACTGT
Sequence is shown in 528-LF：TGCTCTAGTTCTAGGATTGT
Sequence is shown in 528-BIP：
TCAATCTGAATTCCTTTCCAGAACA-CGTTGATACAATTCCAGAGC
Sequence is shown in 528-B3：ACCTCATCCAAATTACGTAGT.
To examine accuracy rate of the detection method that the technology of the present invention is established on diagnostic sensitivity and specificity, using this
Invent the method established and Real-time PCR methods are tested 29 parts of samples altogether simultaneously.29 parts of samples are from domestic 5
Provinces and cities.
Position of the primer in gene is as follows：
Reaction uses 25 μ L reaction systems, and the 25 μ L LAMP reaction systems may include：The μ of genomic DNA 2 of determinand
L, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH4) 2SO4,0.1%Tween20,0.8M glycine betaine
(betaine), 8mM MgSO4, 1.4mM dNTP each, 8U Bst DNA polymerase, primer addition is：
40pmol528-FIP and 528-BIP, 10pmol 528-F3 and 528-B3,20pmol 528-LF.
Reaction result detects
(indicator method) is detected based on calcein color change：Calcein is a kind of amplification indicator, is when negative
Light orange, it is green when positive.
In order to verify the detection sensitivity of above-mentioned detection method, the genomic DNA of carp sample is infected as standard sample using CEV
This, does 10 doubling dilutions, altogether 7 dilution gradients, and be used as negative control using distilled water.
In order to verify the specificity of above-mentioned detection method, with CEV master samples, carp herpesvirusⅡtype, carp herpesviral
III type and frog viral sample genomic DNA are as template, using distilled water as negative control.
In order to verify the result uniformity of the above method and Real-time PCR detection methods, 29 portions of carps and fancy carp are chosen
Sample, extracting genomic DNA are respectively adopted two methods and detected as template.
Then reacted, using nephelometry and indicator method come testing result.
As a result with analysis
Optimum temperature is screened
Optimum temperature screening is carried out to primer, for temperature from 60-67 DEG C, gradient is 1 DEG C, 63 DEG C as can be seen from Figure 1,64
DEG C, 65 DEG C when first reacted, reacted earliest during due to 63 DEG C, so electing optimum temperature as 63 DEG C.
Sensitivity experiments
By the carp edema virus master sample (10 of 10 times of gradient dilutions (totally 7 dilution factors)-1, 10-2, 10-3, 10-4,
10-5, 10-6With 10-7) for genomic templates for reacting, testing result is shown in Fig. 3.Nephelometry is consistent with indicator method experimental result,
Master sample is diluted 100,000 times, the positive can be still detected with the detection method of the present invention, and the whole reaction time is less than
50min。
(Fig. 2 schemes sensitivity Detection result for coloring agent method Sample Negative positive contrast, and Fig. 3 is carp edema virus LAMP
Detection method nephelometry testing result, Fig. 4 are the testing result of coloring agent method sensitivity)：
In Fig. 4 from right to left, template concentrations are followed successively by：10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7With feminine gender pair
According to (distilled water).
Specificity experiments
Using carp edema virus, carp herpesvirusⅡtype, the type of carp herpesviral III and frog viral sample genomic DNA as
Template, using distilled water as negative control, LAMP reactions are carried out, testing result is shown in Fig. 4.As a result show, except carp edema virus-like
Outside product, positive reaction does not occur for other viral samples.Showing the LAMP detection method of the carp edema virus of the present invention has
Higher specificity, it can specifically detect carp edema virus.
Fig. 5 is specific visible color method testing result, is from left to right followed successively by：Carp edema virus, carp herpesviral
II type, the type of carp herpesviral III, frog viral sample result.
Pattern detection result
Detected respectively with 29 portions of carps and fancy carp sample, extracting genomic DNA as template using two methods.Refer to
Show that agent method result is shown in Fig. 6.
By being detected to 29 parts of samples, and compared with fluorescence quantifying PCR method and quality evaluation, institute of the present invention
The method of foundation has good specificity (accuracy rate in specificity is 100%)；Standard on diagnostic sensitivity simultaneously
True rate also more a height of 93.8%；The result coincidence rate of two kinds of detection methods is 96.7%.The foundation of this method, disclosure satisfy that CEV
Big flux, quick detection demand, have a extensive future.
This detection method by calcein by being directly added into reaction system, to result interpretation without beating again since reaction
Reaction tube is opened, the generation for causing false positive because forming aerosol can be effectively prevented from.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (9)
- A kind of 1. detection kit for detecting carp edema virus, it is characterised in that the detection of the detection carp edema virus Kit include respectively the first inner primer containing sequence shown in 528-FIP, 528-BIP, 528-F3,528-B3 and 528-LF and Second inner primer, the first outer primer and the second outer primer and ring primer；Wherein：Sequence is shown in 528-F3：TCCCACAGAATGTAATCTCAASequence is shown in 528-FIP：GTCTGTCTTCGATAAGACAATGACT-TGTGGAGTTTTTGAAGTATACTGTSequence is shown in 528-LF：TGCTCTAGTTCTAGGATTGTSequence is shown in 528-BIP：TCAATCTGAATTCCTTTCCAGAACA-CGTTGATACAATTCCAGAGCSequence is shown in 528-B3：ACCTCATCCAAATTACGTAGT.
- 2. the detection kit of detection carp edema virus as claimed in claim 1, it is characterised in that described detection carp The detection kit of edema virus is reacted for ring mediated constant temperature nucleic acid amplification, and sequence shown in 528-FIP is the first inner primer, Sequence shown in 528-BIP is the second inner primer, and sequence shown in 528-F3 is the first outer primer, and sequence shown in 528-B3 is outside second Primer, sequence shown in 528-LF are ring primer.
- 3. the detection kit of detection carp edema virus as claimed in claim 1, it is characterised in that described detection carp The detection kit of edema virus designs primer for the P4a 528bp genetic fragments of carp edema virus, and target sequence is as follows：ATGGAGTATCCAAAGTACTTAGATTAATGTTATCAATGAAATTTGTGTATTGTGTTTTTGTTAGTCCAAGAGT TTTCTTCTCATCGTTTGTTACTTTTTGTAGTTGCTTAATATTTGTGATAAGATTTCCATTGGCATAAAATCCTTCCC AAATTTGTGTTGAAACATGTTTTAGTGTTTTGTAGATTGTAGCATTTCCTAGTTTGTATGGCAAGAAACAAACTCTC TTTACTGAAACTCCTTGAGGAATTTGATCTAGAATCCCACAGAATGTAATCTCAAATTTGTTTGTGGAGTTTTTGAA GTATACTGTTTCATCATACAATCCTAGAACTAGAGCAAGATTAGAAGTCATTGTCTTATCGAAGACAGACATCTTAT TCCAATCATCAATCTGAATTCCTTTCCAGAACATAACATTTGCAATTTTAACTTGCTCTGGAATTGTATCAACGTAT CCAATATCTTTCTTTACTACGTAATTTGGATGAGGTAGTACTTTGCTAACAAAGTCACAATAGTGAAGAG。
- 4. the detection kit of detection carp edema virus as claimed in claim 1, it is characterised in that described detection carp Sequence to be amplified is divided into seven independent regions by the detection kit of edema virus, is separately designed instead according to this seven regions Answer 5 required primers, including the first inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer.
- 5. the detection kit of detection carp edema virus as claimed in claim 1, it is characterised in that described detection carp The detection kit of edema virus generates Mg during the course of the reaction2P2O7That is magnesium pyrophosphate white precipitate, sentenced by turbidity change The yin and yang attribute of disconnected reaction；Turbidity, which rises, represents carp edema virus, the unchanged expression testing sample of turbidity in testing sample be present In be not present carp edema virus.
- 6. a kind of detection method for detecting carp edema virus, it is characterised in that pass through the P4a 528bp to carp edema virus Genetic fragment designs primer, and target sequence is as follows：ATGGAGTATCCAAAGTACTTAGATTAATGTTATCAATGAAATTTGTGTATTGTGTTTTTGTTAGTCCAAGAGT TTTCTTCTCATCGTTTGTTACTTTTTGTAGTTGCTTAATATTTGTGATAAGATTTCCATTGGCATAAAATCCTTCCC AAATTTGTGTTGAAACATGTTTTAGTGTTTTGTAGATTGTAGCATTTCCTAGTTTGTATGGCAAGAAACAAACTCTC TTTACTGAAACTCCTTGAGGAATTTGATCTAGAATCCCACAGAATGTAATCTCAAATTTGTTTGTGGAGTTTTTGAA GTATACTGTTTCATCATACAATCCTAGAACTAGAGCAAGATTAGAAGTCATTGTCTTATCGAAGACAGACATCTTAT TCCAATCATCAATCTGAATTCCTTTCCAGAACATAACATTTGCAATTTTAACTTGCTCTGGAATTGTATCAACGTAT CCAATATCTTTCTTTACTACGTAATTTGGATGAGGTAGTACTTTGCTAACAAAGTCACAATAGTGAAGAG；Sequence to be amplified is divided into seven independent regions, 5 primers according to needed for this seven regions separately design reaction, Including the first inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer, for specific amplification Target gene；The first described inner primer and the second inner primer, the first outer primer and the second outer primer and ring primer specificity identification target Seven isolated areas in sequence, cause self-loopa strand replacement reaction, at 60~67 DEG C in the presence of Bst Large fragment polymerases In the range of isothermal reaction 60min, synthesize target dna while with accessory substance white magnesium pyrophosphate precipitate produce；With double Steaming water is negative control；The yin and yang attribute of reaction is judged by turbidity change.
- 7. the detection method of detection carp edema virus as claimed in claim 6, it is characterised in that based on calcein color Change detection yin and yang attribute, calcein amplification indicator is added in reaction solution, according to the color change judged result of reaction solution, Green represents carp edema virus in testing sample be present, and carp edema virus is not present in orange expression testing sample.
- 8. the detection method of detection carp edema virus as claimed in claim 6, it is characterised in that the first described inner primer Containing the sequence shown in 528-FIP, the second inner primer contains the sequence shown in 528-BIP, and the first outer primer contains 528-F3 institutes The sequence shown, the second outer primer contain the sequence shown in 528-B3, and described ring primer contains the sequence shown in 528-LF；Its In：Sequence is shown in 528-F3：TCCCACAGAATGTAATCTCAASequence is shown in 528-FIP：GTCTGTCTTCGATAAGACAATGACT-TGTGGAGTTTTTGAAGTATACTGTSequence is shown in 528-LF：TGCTCTAGTTCTAGGATTGTSequence is shown in 528-BIP：TCAATCTGAATTCCTTTCCAGAACA-CGTTGATACAATTCCAGAGCSequence is shown in 528-B3：ACCTCATCCAAATTACGTAGT.
- 9. the detection method of detection carp edema virus as claimed in claim 6, it is characterised in that described isothermal reaction Temperature is 63 DEG C, reaction time 40-60min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897918A (en) * | 2018-08-06 | 2019-06-18 | 北京市水产技术推广站 | Carp edema virus and Koi herpesvirus dual real-time fluorescence quantitative detecting method |
| CN110205407A (en) * | 2019-06-21 | 2019-09-06 | 北京市水产技术推广站 | Quickly the RAA amplimer and probe of detection carp edema virus and detection kit and application method |
| CN110894545A (en) * | 2018-09-13 | 2020-03-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for carp edema disease (CEV) |
| CN112795705A (en) * | 2021-03-12 | 2021-05-14 | 长沙海关技术中心 | Primer and kit for efficient triple detection of SVCV, IHNV and CEV |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006254750A (en) * | 2005-03-16 | 2006-09-28 | Eiken Chem Co Ltd | Method for detecting carp herpes virus (khv) |
| CN102230021A (en) * | 2011-06-03 | 2011-11-02 | 宁波检验检疫科学技术研究院 | Tobacco ringspot virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection reagent and preparation method and use thereof |
| CN102382879A (en) * | 2011-10-21 | 2012-03-21 | 宁波大学 | Pseudomonas fluorescens LAMP (loop-mediated isothermal amplification) detection agent and kit |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
| CN104328209A (en) * | 2014-11-24 | 2015-02-04 | 济南市中心医院 | Primer and kit for fast detection method of leukemia minimal residual disease WT1 gene |
| CN104711351A (en) * | 2015-03-16 | 2015-06-17 | 山东大学附属济南市中心医院 | Specific primer combination for detecting streptococcus pyogenes based on loop-mediated isothermal amplification method and application of specific primer combination |
| CN106399592A (en) * | 2016-12-14 | 2017-02-15 | 通威股份有限公司 | Kit and method for detecting carp edema viruses |
| CN106755568A (en) * | 2016-11-15 | 2017-05-31 | 中华人民共和国东港出入境检验检疫局 | Primer sets and its application for identifying hepatitis A virus |
| CN107058633A (en) * | 2017-06-08 | 2017-08-18 | 浙江省淡水水产研究所 | A kind of carp edema virus visualization quick detection kit and its detection method |
-
2017
- 2017-11-06 CN CN201711075343.4A patent/CN107746898A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006254750A (en) * | 2005-03-16 | 2006-09-28 | Eiken Chem Co Ltd | Method for detecting carp herpes virus (khv) |
| CN102230021A (en) * | 2011-06-03 | 2011-11-02 | 宁波检验检疫科学技术研究院 | Tobacco ringspot virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection reagent and preparation method and use thereof |
| CN102382879A (en) * | 2011-10-21 | 2012-03-21 | 宁波大学 | Pseudomonas fluorescens LAMP (loop-mediated isothermal amplification) detection agent and kit |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
| CN104328209A (en) * | 2014-11-24 | 2015-02-04 | 济南市中心医院 | Primer and kit for fast detection method of leukemia minimal residual disease WT1 gene |
| CN104711351A (en) * | 2015-03-16 | 2015-06-17 | 山东大学附属济南市中心医院 | Specific primer combination for detecting streptococcus pyogenes based on loop-mediated isothermal amplification method and application of specific primer combination |
| CN106755568A (en) * | 2016-11-15 | 2017-05-31 | 中华人民共和国东港出入境检验检疫局 | Primer sets and its application for identifying hepatitis A virus |
| CN106399592A (en) * | 2016-12-14 | 2017-02-15 | 通威股份有限公司 | Kit and method for detecting carp edema viruses |
| CN107058633A (en) * | 2017-06-08 | 2017-08-18 | 浙江省淡水水产研究所 | A kind of carp edema virus visualization quick detection kit and its detection method |
Non-Patent Citations (6)
| Title |
|---|
| HUI ZHANG等: ""A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cyprinid Herpesvirus 2 in Gibel Carp (Carassius auratus gibelio)"", 《THE SCIENTIFIC WORLD JOURNAL》 * |
| M MATRAS等: ""Carp edema virus in Polish aquaculture – evidence of significant sequence divergence and a new lineage in common carp Cyprinus carpio (L.)"", 《JOURNAL OF FISH DISEASES> * |
| 孙颖杰等: "《出入境动物检验检疫技术研究》", 31 July 2009, 中国海洋大学出版社 * |
| 林文慧等: ""多重环介导等温扩增技术研究进展"", 《遗传HEREDITAS》 * |
| 汪川等: "《分子生物学检验技术》", 31 August 2016, 四川大学出版社 * |
| 王小亮等: ""养殖鲤和锦鲤中的鲤浮肿病毒检测与分析"", 《中国畜牧兽医》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897918A (en) * | 2018-08-06 | 2019-06-18 | 北京市水产技术推广站 | Carp edema virus and Koi herpesvirus dual real-time fluorescence quantitative detecting method |
| CN109897918B (en) * | 2018-08-06 | 2022-06-07 | 北京市水产技术推广站 | Double real-time fluorescence quantitative detection method for carp edema virus and koi herpesvirus |
| CN110894545A (en) * | 2018-09-13 | 2020-03-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for carp edema disease (CEV) |
| CN110205407A (en) * | 2019-06-21 | 2019-09-06 | 北京市水产技术推广站 | Quickly the RAA amplimer and probe of detection carp edema virus and detection kit and application method |
| CN112795705A (en) * | 2021-03-12 | 2021-05-14 | 长沙海关技术中心 | Primer and kit for efficient triple detection of SVCV, IHNV and CEV |
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