CN109234452A - A kind of Tilapia mossambica parvovirus TiPV LAMP detection primer and application - Google Patents

A kind of Tilapia mossambica parvovirus TiPV LAMP detection primer and application Download PDF

Info

Publication number
CN109234452A
CN109234452A CN201811121683.0A CN201811121683A CN109234452A CN 109234452 A CN109234452 A CN 109234452A CN 201811121683 A CN201811121683 A CN 201811121683A CN 109234452 A CN109234452 A CN 109234452A
Authority
CN
China
Prior art keywords
parvovirus
tilapia mossambica
tipv
primer
tilapia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811121683.0A
Other languages
Chinese (zh)
Other versions
CN109234452B (en
Inventor
刘文枝
曾令兵
范玉顶
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangtze River Fisheries Research Institute CAFS
Original Assignee
Yangtze River Fisheries Research Institute CAFS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangtze River Fisheries Research Institute CAFS filed Critical Yangtze River Fisheries Research Institute CAFS
Priority to CN201811121683.0A priority Critical patent/CN109234452B/en
Publication of CN109234452A publication Critical patent/CN109234452A/en
Application granted granted Critical
Publication of CN109234452B publication Critical patent/CN109234452B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to field of virus detection, specifically disclose a kind of Tilapia mossambica parvovirus L AMP detection primer and application, inventor has isolated a kind of parvovirus from illness Tilapia mossambica adult fish, the virus is Tilapia mossambica parvovirus (Tilapia parvovirus) TiPV, deposit number are as follows: CCTCC NO:V201856 has ground-breaking meaning for the research of Tilapia mossambica parvovirus.For the virus-specific sequence design primer, Tilapia mossambica parvovirus can be detected, specific good, the high sensitivity of the primer.

Description

A kind of Tilapia mossambica parvovirus TiPV LAMP detection primer and application
Technical field
The invention belongs to viruses molecule detection technique fields, and in particular to a kind of Tilapia mossambica parvovirus TiPV LAMP inspection Survey primer and application.
Technical background
Parvovirus (Parvovirus) is the smallest Single-stranded DNA virus found so far, and virion diameter is The icosahedron of 18~30nm, no cyst membrane, etc. axial symmetry.Parvovirus is extremely wide in distributed in nature, can infect people, vertebra moves Object and invertebrate, and it is related to a variety of diseases.Wherein human parvovirus B19 (Human parvovirus B19, B19) Be known from, can cause a series of serious autoimmune diseases of child, infection in pregnancy will lead to fetus edema, miscarriage or Congenital infection, it is larger to newborn and child's harm, cause worldwide extensive concern.Therefore, to parvovirus into Row further investigation is of great significance.In addition, parvovirus can also cause domestic animal various acute infectious disease, such as canine parvovirus, Pig bocavirus, goose parvovirus disease etc..International Commission on Virus Classification (International Committee on Taxo Nomy of Viruses, ICTV) newest virus taxis report has been delivered in 2014, by Parvoviridae (Pavoviradae) be divided into 8 categories: Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus、Dependo parvovirus、Erythroparvovirus、Protoparvovirus、 Tetraparvovirus.The parvovirus divided at present does not occur the relevant report of infection of marine fishes parvovirus, the application in belonging to Newly identified Tilapia mossambica parvovirus belongs to new virus in patent, the mortality in a short time of China Tilapia mossambica is caused, to me The cultivation and outlet of state Tilapia mossambica bring huge economic loss.
The applicant once separated parvovirus in Tilapia mossambica, but the virus does not make Tilapia mossambica have obvious morbidity disease Shape；And parvovirus disclosed by the invention can cause Tilapia mossambica eyeball, the gill cover, lower jaw and abdomen to have apparent bleeding.Therefore Detection primer based on virus provided by the invention and its design is more for practical significance, to the healthy shape of monitoring cultivating tilapia Condition is particularly important.
Summary of the invention
The object of the present invention is to provide the specific gene of Tilapia mossambica parvovirus TiPV a kind of, the gene is Shown in SEQ ID NO.1, using the primer of the gene and the design of the gene difference of other viruses, it can be used for detecting Tilapia mossambica tiny The deposit number of viral TiPV, the Tilapia mossambica parvovirus TiPV are CCTCC NO:V201856.
It is another object of the present invention to provide the LAMP primer based on sequence design shown in SEQ ID NO.1, institutes The primer stated is F3:5'-CAACTAAAGACCCGGTTCC-3', B3:5'-GCCTTGGTAGCGTAAGTTCA-3'；FIP:5'- GCTATCTCCTCGTTGCTCGGTG-ATCCAGCAATTCCGGAGACA-3', BIP:5'-AGGACGGCCCGGAAGTACTG- TCAAATCCGAGCTGTGTAGC-3'。
Final object of the present invention is the provision of using including polynucleotides shown in SEQ ID NO.1, Huo Zheji The primer of the design of the polynucleotides shown in SEQ ID NO.1 is in the application being prepared into Tilapia mossambica parvovirus detection kit.
In order to achieve the above object, the present invention takes following technical measures:
A kind of specific gene of Tilapia mossambica parvovirus TiPV, the gene are sieve shown in SEQ ID NO.1 The deposit number of non-fish parvovirus TiPV is CCTCC NO:V201856.
The primer of gene difference design based on the gene and other viruses also belongs to protection scope of the present invention.
A kind of Tilapia mossambica parvovirus TiPV LAMP detection primer, including F3:5'-CAACTAAAGACCCGGTTCC-3', B3:5'-GCCTTGGTAGCGTAAGTTCA-3'；FIP:5'- GCTATCTCCTCGTTGCTCGGTG- ATCCAGCAATTCCGGAGACA-3', BIP:5'-AGGACGGCCCGGAAGTACTGTCAAATCCGAGCTGTGTAGC-3'.
A kind of application of sequence shown in Tilapia mossambica parvovirus TiPV LAMP detection primer or SEQ ID NO.1, including benefit It is prepared into above-mentioned primer or sequence for detecting Tilapia mossambica parvovirus detection kit, or is directly used in the tiny disease of Tilapia mossambica The detection of poison.
Compared with prior art, the invention has the following advantages that
1. the present invention has separated the parvovirus with pathogenicity from illness Tilapia mossambica, for Tilapia mossambica parvovirus Research has ground-breaking meaning.
2. method of the invention is fast and convenient: the LAMP method that the present invention is built only needs a few hours, compares more traditional virus Separation method time a couple of days, greatly improve detection efficiency.
3. method high specificity of the invention: the identification in the distinguished sequence area of two pairs of primer pair target sequences ensure that LAMP The high degree of specificity of method amplification.
Specific embodiment
Technical solution of the present invention is if not otherwise specified the conventional scheme of this field, agents useful for same or raw material, If not otherwise specified, it is purchased from commercial channel or discloses.
Embodiment 1:
A kind of Tilapia mossambica parvovirus TiPV, separation process are as follows:
1) will take after the tissues such as viruliferous Tilapia mossambica spleen, kidney take out and mill, culture medium dilutes 100 times, 2000rpm from It takes supernatant to filter after the heart, is inoculated into Tilapia mossambica kidney cell system (TilapiaKidney, TiK) single layer, 25 DEG C are cultivated 7 days, are generated Cytopathic effect (CPE), it is seen that TiK cellular contraction is rounded, diopter increases, and part cell starts shedding off, and cell monolayer is opened Begin to rupture.
Culture medium: M199,10% fetal calf serum, pH 7.0~7.2
2) sick cell is collected, carries out Morphological Identification using transmission electron microscope.
3) virus is collected, sequencing obtains the specific gene of Tilapia mossambica parvovirus TiPV, and the gene is SEQ ID Shown in NO.1.
The morphological feature of Tilapia mossambica parvovirus: virus is spherical in shape, and virus is without cyst membrane, with typical parvovirus form phase Symbol, the virus have been sent to China typical culture collection center on September 20th, 2018 and have carried out preservation, classification naming: Tilapia mossambica Parvovirus (Tilapia parvovirus, TiPV) TiPV, deposit number: CCTCC NO:V201856, address: Wuhan, China Wuhan University.
Embodiment 2:
The difference of specific gene (shown in SEQ ID NO.1) and other viral genes based on Tilapia mossambica parvovirus TiPV The primer of different design:
A kind of Tilapia mossambica parvovirus TiPVLAMP detection primer, including F3:5'-CAACTAAAGACCCGGTTCC-3', B3:5'-GCCTTGGTAGCGTAAGTTCA-3'；FIP:5'- GCTATCTCCTCGTTGCTCGGTG- ATCCAGCAATTCCGGAGACA-3', BIP:5'-AGGACGGCCCGGAAGTACTG-TCAAATCCGAGCTGTGTAGC-3'.
Embodiment 3:
Detection using Tilapia mossambica parvovirus TiPV LAMP detection primer to Tilapia mossambica parvovirus, method include:
1. the extraction of Rofe fish tissues or infection cell total DNA: extracting tissue using Omega (USA) DNA extraction kit DNA。
2.LAMP reaction amplification:
Using 25 μ l reaction systems: it include: each 0.8 μM of inner primer FIP and BIP, each 0.1 μM of outer primer F3 and B3, dNTPs 1mM, Betaine 0.5M, DTT 4mM, MgCl28mM, Bst archaeal dna polymerase 8U, AMV reverse transcriptase 5U, template ribonucleic acid 5 μ l, 1 ×ThermoPol Reaction Buffer.Reaction tube incubates after sixty minutes in 65 DEG C, and 80 DEG C inactivate 2 minutes.
F3:5'-CAACTAAAGACCCGGTTCC-3',
B3:5'-GCCTTGGTAGCGTAAGTTCA-3'；
FIP:5'- GCTATCTCCTCGTTGCTCGGTG-ATCCAGCAATTCCGGAGACA-3',
BIP:5'-AGGACGGCCCGGAAGTACTG-TCAAATCCGAGCTGTGTAGC-3'.
LAMP reacts amplified production analysis:
1. taking 5 μ L amplified productions to be placed in gel imaging system and be imaged after 2% agarose gel electrophoresis, electrophoretogram Piece shows scalariform LAMP signature band, and result is the positive；If result is feminine gender without any band.
2. 1000 × SYBR Green I, 2 μ L is added into reaction tube after reaction, knot is observed after standing 1~5min Fruit, reaction solution greening are then the positive, and keeping orange is then feminine gender.
Embodiment 4:
Tilapia mossambica parvovirus L AMP detection primer specific test
Using method described in embodiment 3, the viral sample to be checked in table 1 is detected, while negative control is set Group (distilled water) and positive controls (TiPV), viral sample to be checked specifically: giant salamander irido virus is viral (GSIV), carp bleb Virus Type II (CyHV-2), Koi herpesvirus (KHV), carp edema viral (CEV) and Tilapia mossambica Luohu are viral (TiLV).
1 Tilapia mossambica parvovirus L AMP detection kit specific test of table
Test number Title Amplification
1 TiPV +
2 CyHV-2 ?
3 KHV ?
4 CEV ?
5 TiLV ?
The results are shown in Table 1, and only trapezoidal characteristics band can be detected in positive controls (TiPV), remaining group is without feature Band illustrates that Tilapia mossambica parvovirus TiPV LAMP detection primer has very strong specificity.
Embodiment 5:
The sensitivity of Tilapia mossambica parvovirus L AMP detection primer
The 10 times of gradient dilutions of TiPVDNA template for being 2100ng/ μ L by concentration, using the method for embodiment 3, detection is utilized The detection of primer LAMP provided by the invention limits, the results show that 105Purpose band also can be detected in dilution again, that is, minimum The DNA profiling of detectable 21.00pg/ μ L.
Embodiment 6:
Application of the Tilapia mossambica parvovirus TiPVLAMP detection primer in preparation Tilapia mossambica parvovirus detection kit:
1. the extraction of Rofe fish tissues total DNA: extracting infection Tilapia mossambica parvovirus using Omega (USA) kit The total DNA of the viscera tissues such as the spleen kidney of the Tilapia mossambica of TiPV is saved backup obtaining -20 DEG C of DNA profiling.
2.LAMP reaction amplification:
Using 25 μ l reaction systems: it include: each 0.8 μM of inner primer FIP and BIP, each 0.1 μM of outer primer F3 and B3, dNTPs 1mM, Betaine 0.5M, DTT 4mM, MgCl28mM, Bst archaeal dna polymerase 8U, AMV reverse transcriptase 5U, template ribonucleic acid 5 μ l, 1 ×ThermoPol Reaction Buffer.Reaction tube incubates after sixty minutes in 65 DEG C, and 80 DEG C inactivate 2 minutes.
F3:5'-CAACTAAAGACCCGGTTCC-3', B3:5'-GCCTTGGTAGCGTAAGTTCA-3'；FIP:5'- GCTATCTCCTCGTTGCTCGGTG-ATCCAGCAATTCCGGAGACA-3', BIP:5'-AGGACGGCCCGGAAGTACTG- TCAAATCCGAGCTGTGTAGC-3'。
3.LAMP reacts amplified production analysis:
1. taking 5 μ L amplified productions to be placed in gel imaging system and be imaged after 2% agarose gel electrophoresis, electrophoretogram Piece shows scalariform LAMP signature band, and result is the positive；If result is feminine gender without any band.
2. 1000 × SYBR Green I, 2 μ L is added into reaction tube after reaction, knot is observed after standing 1~5min Fruit, reaction solution greening are then the positive, and keeping orange is then feminine gender.
The results show that positive control (Tilapia mossambica parvovirus TiPV) shows trapezoidal characteristics band, negative control (water) Without obvious band, No. 1-5 Tilapia mossambica tissue sample for having infected Tilapia mossambica parvovirus TiPV shows apparent trapezoidal characteristics Band illustrates that sample 1-5 is that Tilapia mossambica parvovirus is positive, is consistent with actual conditions.
Sequence table
<110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120>a kind of Tilapia mossambica parvovirus TiPV LAMP detection primer and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 224
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caactcaaga cccggttccg agcacggatc cagcaattcc ggagacagaa ttcagatcgg 60
ggacattgga gaattggttt ctggatacac cgagcaacga ggagatagcc gagcagtttt 120
cgggggagga cggcccggaa gtactgaact atttgagagc gaggaacacc cttttctttt 180
ggctacacag ctcggatttg agtctgaact tacgctacca aggc 224
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
caactaaaga cccggttcc 19
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gccttggtag cgtaagttca 20
<210> 4
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gctatctcct cgttgctcgg tgatccagca attccggaga ca 42
<210> 5
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aggacggccc ggaagtactg tcaaatccga gctgtgtagc 40

Claims (4)

1. a kind of Tilapia mossambica parvovirus (Tilapia parvovirus) TiPV specific gene, the gene is SEQ ID Shown in NO.1, the deposit number of Tilapia mossambica parvovirus (Tilapia parvovirus) TiPV are as follows: CCTCC NO: V201856。
2. the primer based on specific gene described in claim 1 design.
3. primer according to claim 2, the primer are as follows: F3:5'-CAACTAAAGACCCGGTTCC-3', B3: 5'-GCCTTGGTAGCGTAAGTTCA-3'；FIP:5'- GCTATCTCCTCGTTGCTCGGTG- ATCCAGCAATTCCGGAGACA-3', BIP:5'-AGGACGGCCCGGAAGTACTG-TCAAATCCGAGCTGTGTAGC-3'.
4. gene described in claim 1 or primer as claimed in claim 2 are in preparation Tilapia mossambica parvovirus detection kit In application.
CN201811121683.0A 2018-09-26 2018-09-26 TiPV LAMP (Loop-mediated isothermal amplification) detection primer for tilapia parvovirus and application Active CN109234452B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811121683.0A CN109234452B (en) 2018-09-26 2018-09-26 TiPV LAMP (Loop-mediated isothermal amplification) detection primer for tilapia parvovirus and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811121683.0A CN109234452B (en) 2018-09-26 2018-09-26 TiPV LAMP (Loop-mediated isothermal amplification) detection primer for tilapia parvovirus and application

Publications (2)

Publication Number Publication Date
CN109234452A true CN109234452A (en) 2019-01-18
CN109234452B CN109234452B (en) 2020-06-19

Family

ID=65057739

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811121683.0A Active CN109234452B (en) 2018-09-26 2018-09-26 TiPV LAMP (Loop-mediated isothermal amplification) detection primer for tilapia parvovirus and application

Country Status (1)

Country Link
CN (1) CN109234452B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1946733A (en) * 2004-02-06 2007-04-11 高级生物营养公司 RNA-mediated interference to control disease in terrestrial and aquaculture animals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1946733A (en) * 2004-02-06 2007-04-11 高级生物营养公司 RNA-mediated interference to control disease in terrestrial and aquaculture animals

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIANG DU ET AL.: "Identification of a Novel Ichthyic Parvovirus in Marine Species in Hainan Island, China", 《FRONTIERS IN MICROBIOLOGY》 *
LIU,W. ET AL.: "MN584742.1", 《GENBANK》 *
马杰等: "罗非鱼暴发性流行病病毒病原的电镜观察初报", 《淡水渔业》 *

Also Published As

Publication number Publication date
CN109234452B (en) 2020-06-19

Similar Documents

Publication Publication Date Title
CN109439634B (en) Pseudorabies virus gene engineering attenuated vaccine strain and application thereof
CN109517806B (en) Tilapia lake virus virulent strain and RPA detection primer and method thereof
CN106591242B (en) One plant of canine parvovirus poison strain CPV-YH and its application
CN102703608A (en) Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses
CN109234450A (en) A kind of swamp eel rhabdovirus CrERV RT-CPA detection primer and application
CN107201345B (en) A kind of crucian coronavirus HB93 and RT PCR detection primers and application
CN103205511B (en) Primer pair for detecting pigeon torque teno viruses and application of primer pair
CN105821163B (en) A kind of dual-PCR method of Rapid identification goose parvovirus and cherry valley duck source parvovirus
CN103911461A (en) A method of simultaneously detecting a plurality of garlic viruses by quadruple RT-PCR and a primer composition thereof
CN109182277B (en) Monopterus albus rhabdovirus CrERV and RT-PCR detection primer and application
CN112899404B (en) Nested-RAA detection primer pair for mandarin fish frog iridovirus as well as application and detection method thereof
CN111118215A (en) Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting capripoxvirus virus
CN109234451A (en) A kind of Tilapia mossambica parvovirus TiPV CPA detection primer and application
CN112301168B (en) TaqMan real-time fluorescent quantitative RT-PCR kit and method for detecting largemouth black bass double RNA virus
CN104928260A (en) Infectious bovine rhinotracheitis virus IBRV-JN03 isolate and application thereof
CN106148566B (en) A kind of giant salamander irido virus CPA detection primer and application
CN109266785B (en) Monopterus albus rhabdovirus CrERV RT-LAMP detection primer and application
CN108060265A (en) For detecting the primer sets of the oyster herpetovirus of infection blood clam and probe and its application
CN109234452A (en) A kind of Tilapia mossambica parvovirus TiPV LAMP detection primer and application
CN115044556B (en) Carp brain cell line and application thereof
Khoo et al. First report of Epicoccum sorghinum causing leaf spot on Platostoma palustre in Malaysia
CN104630153A (en) Porcine pseudorabies virus strain and application thereof
CN108707589B (en) Bovine viral diarrhea virus SMU-Z6/1a/SC/2016 isolate and application thereof
CN109234243A (en) A kind of Tilapia mossambica parvovirus TiPV and PCR detection primer and application
CN102628088B (en) Polymerase chain reaction (PCR) detection method universal for viruses

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant