CN107190103A - Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously - Google Patents
Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The present invention is provided to detect common eel herpesviral EHV, Japanese eel epidermal cell necrosis virus JEECV and Anguilla marmorata polyomavirus AmPV multiple PCR primer group, kit and detection method simultaneously, nucleotide sequence of the invention according to acquisition, designed for amplification EHV, JEECV and AmPV specific primer, avoid forming stable primer dimer between primer, and the specificity of primer extension increasing sequence is analyzed, acquisition is made up of 6 nucleotide sequences, can be simultaneously to EHV, JEECV and AmPV have high amplification sensitivity and specific primer sets, including：SEQ ID NO：1‑6.The present invention can simultaneously detect in same PCR reaction systems and differentiate EHV, JEECV and AmPV, with easy to operate, the advantages of high specificity；And the present invention can according to amplification length difference directly result of determination, more efficient, practicality.
Description
Technical field
The invention belongs to aquatic products disease technical field of microbial detection, and in particular to one kind can detect common eel blister sore simultaneously
Malicious (Eel herpesvirus, EHV), Japanese eel epidermal cell necrosis virus (Japanese eel endothelial
Cells-infecting virus, JEECV) and Anguilla marmorata polyomavirus (Anguilla marmorata polyoma-like
Virus, AmPV) three kinds of DNA virus multiple PCR primer group, kit and detection method.
Background technology
China is one of main common eel cultivation country of the world, and its output value occupies weight in the export trade in aquatic products of China
Want status.The main Japanese eel of kind (A.japonica), European eel (A. anguilla), the America common eel of cultivation
(A.rostrata), Anguilla marmorata (A.Marmorata).Due to large-scale intensive culture, great viral blight breaks out day
It is beneficial frequent, cause huge economic loss.The infective pathogen for specifying disease is effective important prerequisite for carrying out control and prevention of disease.I
Take the lead in establishing the detection methods of a variety of common eels viruses at home, carried out the epidemiology survey of correlated virus.Common eel blister
Exanthema virus (Eel herpesvirus, EHV), Japanese eel endothelial cell necrosis virus (Japanese eel
Endothelial cells-infecting virus, JEECV) and Anguilla marmorata polyomavirus (Anguilla marmorata
Polyoma-like virus, AmPV) DNA virus is belonged to, these three viruses show as gill rot, gone out to the pathogenic of common eel
The symptoms such as blood, are difficult to differentiate between in clinical diagnosis.We establish these three viral PCR detection methods respectively, detect respectively
Time and effort consuming；Meanwhile, also repeatedly find that they have the situation of mixed infection.
EHV is a kind of double-stranded DNA virus with cyst membrane, and once the cultivation dealer to multinational European eel and Japanese eel made
Into huge economic losses, it is also the key factor for causing wild European eel reduction.We show in the research of early stage, and it can be
Detected on European eel, Japanese eel, America common eel and Anguilla marmorata.Clinical and pathological research shows that EHV can cause " rotten
The classical symptom such as the gill ", " unsticking ", " reddish tone ".But existing PCR detections are all the DNA polymerase gene sequences Designs according to EHV
Primer, because DNA polymerase gene is highly conserved, may detect common eel irido virus in detection, show as EHV false positives.
JEECV is a kind of double-stranded cyclic DNA virus, with the large T antigen DNA homolog with polyomavirus and highly conserved
LTLG genes, be Japanese eel endothelial cell necrosis disease (Viral endothelial cell necrosis of eel,
VECNE infective pathogen).From the eighties in last century, VECNE just often outbursts, the cultivation dealer to Japanese eel causes huge
Economic loss.The fin ray that its pathogenic classical symptom includes common eel reddens, abdominal distention, the gill, liver hyperemia etc..We are at home
JEECV PCR detection methods are established first, and are successfully applied to the diagnosis of disease.
AmPV is as JEECV, the LTLG genes with polyomavirus.Sequence analysis shows, we are from ill flower eel
The AmPV that the sequence expanded in eel is separated with TaiWan, China has larger difference, and homology is 94%.AmPV can cause Anguilla marmorata
The classical symptoms such as gill rot, bleeding of skin.
Therefore, a kind of these three viral multiple PCR primer groups of detection of offer are needed badly, invention detection kit and optimization are real
Condition is tested, realizes and is detected while these three are viral.
The content of the invention
It is an object of the invention to provide a kind of convenient and swift, high specificity, accuracy rate is high and can detect common eel blister simultaneously
Exanthema virus (Eel herpesvirus, EHV), Japanese eel epidermal cell necrosis virus (Japanese eel endothelial
Cells-infecting virus, JEECV) and Anguilla marmorata polyomavirus (Anguilla marmorata polyoma-like
Virus, AmPV) multi-PCR detection method.
To achieve the above object, the present invention provide one kind in same PCR reaction systems simultaneously detect EHV, JEECV and
AmPV multiple PCR primer group and multiple PCR detection kit, and be achieved through the following technical solutions：
The present invention is according to the nucleotide sequence of acquisition, and the specific primer designed for expanding EHV, JEECV and AmPV is kept away
Exempt to form stable primer dimer between primer, and the specificity of primer extension increasing sequence is analyzed, obtain by 6 nucleic acid
Sequence is constituted, and can have high amplification sensitivity and specific primer sets to EHV, JEECV and AmPV simultaneously, including：SEQ
ID NO：1-6；Specifically, can be divided into by being used for that the nucleotide sequence shown in SEQ ID No.1, SEQ ID No.2 is constituted
Detect EHV primer sets 1, by the nucleotide sequence shown in SEQ ID No.3, SEQ ID No.4 constitute be used for detect
JEECV primer sets 2 and by the nucleotide sequence shown in SEQ ID No.5, SEQ ID No.6 constitute be used for detect AmPV's
Primer sets 3 are constituted.
The specific detection result of primer pair shows that the PCR reaction systems specificity is good.Detect EHV 1 pair of brocade of primer sets
The homologys such as carp herpesviral (Koi herpesvirus, KHV), common eel irido virus (Eel iridovirus, EIV) are nearer
Virus without non-specific amplification, detect that JEECV primer sets 2, without specific amplification, detect AmPV 3 pairs of primer sets to AmPV
JEECV is without specific amplification.
The sensitivity result of the test of primer pair shows that the multi-PCR detection method can detect minimum 10 copies
EHV, JEECV and AmPV.
EHV, JEECV and AmPV multiple PCR detection kit include the primer sets that above-mentioned 6 nucleotide sequences are constituted, and PCR delays
Fliud flushing, dNTP, Taq archaeal dna polymerases, ddH2O and positive control dna plasmid composition.Wherein, positive control plasmid is connection
EHV, JEECV and the AmPV of pMD19-T carriers extension increasing sequence, concentration are 108 copies/μ L, and the DNA sequence dna of amplification is as follows：
EHV plasmids pMD19-EHV insetion sequence is SEQ ID No.7；
JEECV plasmids pMD19-JEECV insetion sequence is SEQ ID No.8；
AmPV plasmids pMD19-AmPV insetion sequence is SEQ ID No.9.
EHV, JEECV and the AmPV method of multiplex PCR detection are carried out with above-mentioned detection primer group and detection kit such as
Under：
Prepare testing sample DNA profiling
Extracted using tissue gene group DNA extraction kit or virus genom DNA extracts kit in measuring samples
Genomic DNA.
PCR is detected
Take the sample DNA extracted in the 2 μ L above methods as pcr template, PCR reaction systems are formulated as follows on ice：
It is placed in PCR instrument and enters performing PCR amplification, amplification condition is：
Pre-degeneration：94 DEG C 3 minutes；
Circulation：94 DEG C are denatured 10 seconds, and 60 DEG C are annealed 15 seconds, and 72 DEG C extend 45 seconds, 40 circulations；
Extension：72 DEG C 5 minutes；
Result judgement
10 μ L PCR primers are taken to carry out electrophoresis with 1% agarose gel electrophoresis, if electrophoresis result has in 240bp positions
Amplified band, represents there is EHV in pathological material of disease；If electrophoresis result has amplified band in 505bp positions, represent have in pathological material of disease
JEECV；If electrophoresis result has amplified band in 793bp positions, represent that there is AmPV in pathological material of disease.If electrophoresis result goes out simultaneously
Now many band, represent there is the mixed infection of virus corresponding with stripe size in pathological material of disease.If the equal nothing in position at above-mentioned three
It can be seen that amplified band, is represented in pathological material of disease without above-mentioned three kinds of virus.
Detection common eel herpesviral EHV, Japanese eel epidermal cell necrosis simultaneously are being prepared present invention additionally comprises primer sets
Application in viral JEECV and Anguilla marmorata polyomavirus AmPV product.Described product includes being used to detect three kinds of fish diseases
Kit, gene probe, genetic chip of poison etc..Wherein, gene probe, the preparation method of genetic chip refer to existing skill
Art.
Multiple PCR detection primer group, detection kit and detection method that the present invention is provided, can be in same PCR reactants
Detect simultaneously in system and differentiate EHV, JEECV and AmPV, with easy to operate, the advantages of high specificity；And the present invention can root
According to the difference directly result of determination, more efficient, practicality of amplification length.
Brief description of the drawings
Fig. 1 is the electrophoretogram that specific amplification identification is carried out using primer sets 1, primer sets 2, primer sets 3. M：DL2000
Standard molecular weight Marker；1：EHV；2：KHV；3：EIV；4：JEECV； 5：AmPV；6：AmPV；7：JEECV；
Fig. 2 is to utilize primer sets and kit identification EHV, JEECV, AmPV electrophoretogram.M：DL2000 standard molecular weights
Marker；1：EHV；2：JEECV；3：AmPV；
Fig. 3 is to utilize primer sets and kit identification EHV, JEECV, AmPV sensitivity electrophoretogram.M：DL2000 standards
Molecular weight Marker；1：108copies；2：107copies；3： 106copies；4：105copies；5：104copies；6：
103copies；7：102copies；8： 100copies；9：Negative control；
Fig. 4 is to utilize above-mentioned primer sets and kit identification EHV, JEECV, the electrophoretogram of AmPV pathological material of diseases.M：DL2000 is marked
Quasi-molecule amount Marker；1：Positive control；2：EHV and JEECV mixed infections；3：EHV and AmPV mixed infections；4：EHV；5：
JEECV；6：AmPV；7：Pathological material of disease is not detected；8：Negative control；
Embodiment
To describe the technology contents of the present invention in detail, feature, the objects and the effects being constructed, below in conjunction with embodiment
And coordinate accompanying drawing to be explained in detail.
(1) design and optimization of primer
Cell separation, 8 plants of EHV are identified, have cloned the open reading frame sequence of 6 genes such as EHV ORF8, ORF95,
Its homology is analyzed, 6 pairs of primers in conserved regions design, the size of amplified production is set in about 250bp.Trained with cell separation
Foster EHV DNA are template, enter performing PCR amplification in conventional PCR methods, gel electrophoresis analysis are carried out to amplified production.Screening
Go out the high primer sets 1 of amplification efficiency.
Cell separation, the positive pathological material of diseases of 3 parts of JEECV are identified, cloned JEECV LTLG open reading frame sequence, analyzed
Its homology, 2 pairs of primers are devised in conserved regions design, and the size of amplified production is set in about 500bp.With the positive diseases of JEECV
The genomic DNA extracted in material is template, enters performing PCR amplification with conventional PCR method, gel electrophoresis point is carried out to amplified production
Analysis.Filter out the high primer sets 2 of amplification efficiency.
Cell separation, the positive pathological material of diseases of 5 parts of AmPV are identified, cloned AmPV LTLG open reading frame sequence, analyzed it
Homology, using software Primer Primer 5.0 in conserved regions design 2 pairs of primers, the size of amplified production is set in about
750bp.The genomic DNA extracted using in the positive pathological material of diseases of AmPV enters performing PCR amplification, to expanding as template with conventional PCR method
Increase production thing and carry out gel electrophoresis analysis.Filter out the high primer sets 3 of amplification efficiency.
(2) in pathological material of disease genomic DNA extraction
The doubtful viral disease pathological material of disease of common eel is collected, the viscera tissues such as liver,spleen,kidney are taken, is extracted and tried with tissue DNA after homogenate
Agent box extracts genomic DNA.
(3) specificity of multiplex PCR detection
It is template detection primer with KHV, the EIV higher with EHV homologys to detect the specificity of above-mentioned primer sets amplification
Group 1 expands EHV specificity；Because JEECV and AmPV affiliation is near, and have no the report of other fish polyomavirus,
Therefore the genomic DNA extracted respectively using in the positive pathological material of diseases of JEECV and AmPV expands JEECV spy as template detection primer sets 2
The opposite sex and primer sets 3 expand AmPV specificity.As a result show, detection EHV primer sets 1 are only capable of specific amplification EHV, no
KHV, EIV can be expanded；Detection JEECV primer sets 2 are only capable of specific amplification JEECV, it is impossible to expand AmPV；Detection AmPV's draws
Thing group 3 is only capable of specific amplification AmPV, it is impossible to expand JEECV (as shown in Figure 1).
(4) foundation of multi-PCR detection method
By primer sets 1, primer sets 2, primer sets 3 according to 1：1：1 ratio mixing, final concentration is 10 μM.Moving back respectively
Enter performing PCR amplification under the conditions of fiery temperature 55,60,65, then by amplified conditions optimization, set up following multi-PCR detection method：
10XPCR buffer(Mg2+plus)5μL、dNTP (2.5mM each)4μL、Taq DNA Polymerase(5U/μL)0.5μ
L, the μ L of primer sets 2, supplement ddH2O to 50 μ L.It is placed in PCR instrument and enters performing PCR amplification, amplification condition is：Pre-degeneration：94 pre-degenerations：
94 DEG C 3 minutes；Circulation：94 DEG C are denatured 10 seconds, and 60 DEG C are annealed 15 seconds, and 72 DEG C extend 45 seconds, 40 circulations；Extension：72 DEG C 5 points
Clock.10 μ L PCR primers are taken, through 1% agarose gel electrophoresis, are taken pictures in gel imaging system observation.The inspection set up with the present invention
Survey method is detected to cell culture EHV and the positive pathological material of disease of JEECV, AmPV, as a result can efficiently be detected corresponding virus, not sent out
Existing non-specificity band (as shown in Figure 2).
(5) sensitivity of multiplex PCR
EHV, JEECV, AmPV sequence expanded with primer sets 1, primer sets 2, primer sets 3 is connected to cloning vector respectively
After pMD19-T, sequence verification, DNA content is determined, copy number is calculated, then by three kinds of plasmids according to copy number 1：1：1 mixing,
Concentration is 108 copies/μ L, is used as the positive control of kit.Positive control is subjected to 10 times of gradient dilution, it is many with foundation
Weight PCR detection method is detected.As a result show, this method specific can detect EHV, JEECV, AmPV of minimum 10 copies
(as shown in Figure 3).
(6) the positive common eel pathological material of disease of multiplex PCR detection
To verify the validity of above-mentioned multi-PCR detection method, selection with conventional PCR method detect EHV, JEECV,
Positive AmPV pathological material of disease, mixed infection pathological material of disease and pathological material of disease is not detected, pathological material of disease is detected with above-mentioned multi-PCR detection method,
The positive pathological material of diseases of detection EHV, JEECV, AmPV, and EHV and JEECV, the positive pathological material of disease of EHV and AmPV mixed infections respectively, as a result
It is completely the same (as shown in Figure 4) with the result that is detected with Standard PCR method.
Although the various embodiments described above are described, those skilled in the art once know basic wound
The property made concept, then can make other change and modification to these embodiments, so embodiments of the invention are the foregoing is only,
Not thereby the scope of patent protection of the present invention, the equivalent structure that every utilization description of the invention and accompanying drawing content are made are limited
Or equivalent flow conversion, or other related technical fields are directly or indirectly used in, similarly it is included in the patent of the present invention
Within protection domain.
SEQUENCE LISTING
<110>Fujian Province Agriculture Science Academy, Institute of Biotechnology
<120>Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously
<130> 2017
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
actctggctc gcaaccaatc t 21
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
ccaccaaaca acccagcaca atc 23
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
cccaccagag gaaagacaaa gac 23
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
caccgggttt gtctcatctt cca 23
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
ataggttcgt gctgtccttc aa 22
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence
<400> 6
aaagggactg ccacctgaga tt 22
<210> 7
<211> 240
<212> DNA
<213>Artificial sequence
<400> 7
actctggctc gcaaccaatc tcagtctgtc agagattgtg gtcggagaaa attgcagcgg 60
tgtggactgg aaacaggtgc aaggagtgtg gtgcaattca acatactgtc cccagatgat 120
gagatcggaa gatgtggagt ctatgaggtc cgccatcgtc cacgttctgg agtcagagtc 180
gccgtttgac aagcagttca agtttggatc ctacgtgact ctgattgtgc tgggttgttt 240
<210> 8
<211> 505
<212> DNA
<213>Artificial sequence
<400> 8
cccaccagag gaaagacaaa gactggagca aaggctgaaa aggataaatg aggtatgggc 60
aaaatacctg gacatccatc agagacaaca gacgtggttt gacatatccc ccagtaaaag 120
ggggggcacg ctgactgtac ctgaatacct ggctaaatat tgcactacta tacaggatgg 180
tcagtttgtg cagactgtac tggtgcaggt gccctgggct aagctgggat cagtggtcac 240
agaactgaaa aaatacaaac atgtagatct aattgcaggc ggggaccccc gggaggaccc 300
cccaacaggc atagcagttg tgcaatttgt gcataggcaa aaggaatctg cattgaaggg 360
aaaatgcaat gctgtgacac ttgtgtgcaa tgtgatgcag gtagcaaata aatcatttac 420
tactgttaag caaatatgcc tagcgtattg gaaggaaaat accacagagc acggtcacgt 480
gatggaagat gagacaaacc cggtg 505
<210> 9
<211> 793
<212> DNA
<213>Artificial sequence
<400> 9
atagggttcg tgctgtcctt caaaaggtca tctaggattt cggacagtct ttccatgtaa 60
aattcctgct cagttattgt ttggtatttc aacctttgat gcgccatcac tgttacagtc 120
gcattggcag ctaaacgttt cttgtccttt ttgcagctga aattgctgtg cattgttgaa 180
atgagtttga tgcaaattgt aatggctttc tgggtttctg acctcctcct gtgagggctc 240
tacacatcta gagcagtcgt caggtttgat tgctaattta tcatagttgc ctatgacagc 300
caggcagtct gtgaacccaa acaccatagc aaactcattt aacaacttaa tgctgaacgc 360
attctcgttt gcatcaccct cactgtcatt gaattcatga gactcagcga tggatggctg 420
actacagcga tcatgcaccg gctgacacct tagcctcagt ttgtgaactg attgggccca 480
cttcttggca agaacaaccc gtaccaggca gtgattgaca gatgtaaaca aagttttaag 540
cacattccga agcctagttt gcctatgagg actttgaaat cgcacaagtt gtacgaacat 600
gttgctatca tactcttcat aacaccctgt gacagtaccc tccacatccc ccacacttcc 660
caatgcagcc tgtacaacat catatttgca cctggaataa aacagcagaa gacaatgaac 720
agtgctgtgt gtgttcaaag ccttactcaa aaagtcctga aacaaaggag gaatctcagg 780
tggcagtccc ttt 793
Claims (4)
1. one kind is used to detect that common eel herpesviral EHV, Japanese eel epidermal cell necrosis virus JEECV and Anguilla marmorata are more simultaneously
Tumor virus AmPV multiple PCR primer group, it is characterised in that the sequence of primer is respectively SEQ ID NO in described primer sets：
1-6。
2. the primer sets described in claim 1 are preparing detection common eel herpesviral EHV, Japanese eel epidermal cell necrosis simultaneously
Application in viral JEECV and Anguilla marmorata polyomavirus AmPV product.
3. one kind is used to detect that common eel herpesviral EHV, Japanese eel epidermal cell necrosis virus JEECV and Anguilla marmorata are more simultaneously
Tumor virus AmPV multiple PCR detection kit, including：10 × PCR buffer solutions, dNTPs mixed solutions, primer sets, Taq DNA
Polymerase and sterile deionized water, and can as positive control DNA plasmid, it is characterised in that：The detection primer group is power
Profit requires the multiple PCR primer group described in 1.
4. a kind of detect common eel herpesviral EHV, Japanese eel epidermal cell necrosis virus JEECV and Anguilla marmorata polyoma disease simultaneously
Malicious AmPV method, it is characterised in that
1) virus genom DNA to be detected is extracted from testing sample；
2) multiplexed PCR amplification and product demarcation are carried out；
Include Mg in 50 μ L PCR reaction system2+Concentration is 15mmol/L 10XPCR buffer 5 μ L, 2.5mM/L
The μ L of dNTPs 4, Taq enzyme 0.5 μ L, 10 μM/L the μ L of primer sets 2, step 1) in extract the μ of virus genom DNA to be detected 2
L supplements ddH as template2O to 50 μ L；
PCR amplification conditions are：94 DEG C be denatured 3 minutes, 94 DEG C 10 seconds, 60 DEG C 15 seconds, 72 DEG C 45 seconds, 40 circulation, 72
DEG C extension 5 minutes；
3) result judgement：Take 10 μ L steps 2) in pcr amplification product electrophoresis, wherein common eel blister are carried out with 1% Ago-Gel
Exanthema virus EHV positive bands are located at 240bp；Japanese eel epidermal cell necrosis virus JEEC positive bands are located at 505bp；Flower eel
Eel polyomavirus AmPV positive bands are located at 793bp；If being negative for correspondence Viral diagnosis without correspondence band；If electrophoresis knot
There are the multiple bands consistent with correspondence virus-positive stripe size simultaneously in fruit, then is determined as the coinfection of correspondence virus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315487A (en) * | 2018-04-16 | 2018-07-24 | 福建省农业科学院生物技术研究所 | A kind of primer sets, kit and its application of detection common eel herpesviral |
| CN110863067A (en) * | 2019-11-29 | 2020-03-06 | 厦门海关技术中心 | Primer pair and kit for detecting eel herpes virus |
| CN112852987A (en) * | 2021-03-16 | 2021-05-28 | 青岛农业大学 | PCR (polymerase chain reaction) specific primer and detection method for detecting eel dermatophytosis |
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| JP2008141988A (en) * | 2006-12-07 | 2008-06-26 | Kagoshima Univ | Anguilla fluorescent protein |
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| CN110863067A (en) * | 2019-11-29 | 2020-03-06 | 厦门海关技术中心 | Primer pair and kit for detecting eel herpes virus |
| CN112852987A (en) * | 2021-03-16 | 2021-05-28 | 青岛农业大学 | PCR (polymerase chain reaction) specific primer and detection method for detecting eel dermatophytosis |
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