CN109234451B - Tilapia parvovirus TiPV CPA detection primer and application thereof - Google Patents
Tilapia parvovirus TiPV CPA detection primer and application thereof Download PDFInfo
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- CN109234451B CN109234451B CN201811121682.6A CN201811121682A CN109234451B CN 109234451 B CN109234451 B CN 109234451B CN 201811121682 A CN201811121682 A CN 201811121682A CN 109234451 B CN109234451 B CN 109234451B
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- 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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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14311—Parvovirus, e.g. minute virus of mice
- C12N2750/14322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
The invention belongs to the field of virus detection, and particularly discloses Tilapia parvovirus (Tilapia parvovirus) TiPV CPA detection primers and application thereof, wherein an inventor separates a parvovirus from diseased adult Tilapia for the first time, the virus is TiPV of Tilapia parvovirus (Tilapia parvovirus), and the preservation number is as follows: CCTCC NO: v201856, has pioneering significance for the research of tilapia parvovirus. The primer is designed aiming at the virus sequence, can detect the tilapia parvovirus, and has good specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of virus molecule detection, and particularly relates to TiPV CPA detection primers for tilapia parvovirus and application thereof.
Technical Field
Parvovirus (Parvovirus) is the smallest single-stranded DNA virus found so far, and has an icosahedron, a non-enveloped and equiaxial symmetry, and the diameter of the virus particle is 18-30 nm. Parvoviruses are distributed very widely in nature, can infect humans, vertebrates and invertebrates, and are associated with a variety of diseases. Among them, Human parvovirus B19(Human paroviruses B19, B19) is well known, can cause a series of serious autoimmune diseases of infants, and the pregnancy infection can cause fetal edema, abortion or congenital infection, so that the Human parvovirus has great harm to newborns and infants, thereby causing wide attention worldwide. Therefore, the intensive study of parvovirus is of great significance. In addition, parvovirus can also cause various acute infectious diseases of livestock, such as canine parvovirus, porcine bocavirus, goose parvovirus and the like. The International Committee for Classification of Viruses (International Committee on Taxo nomy of Viruses, ICTV) published a recent report of classification of Viruses in 2014, which classified the parvoviridae (Pavoviradae) into 8 genera, Amdoparvorous, Aveparvorvus, Bocaparvorous, Copiparvorous, Dependo paravorous, Erythroparvorous, Protoparvorous, Tetraparvorous. Related reports of fish infection parvovirus do not appear in the currently divided parvovirus genera, and the newly identified tilapia parvovirus in the application patent belongs to a new virus, so that a large number of tilapia in China die in a short time, and huge economic losses are brought to tilapia culture and export in China.
Parvovirus has been isolated in tilapia by the applicant, but the parvovirus does not cause tilapia to have obvious disease symptoms; the parvovirus disclosed by the invention can cause tilapia eyeball, gill cover, lower jaw and abdomen to have obvious bleeding symptoms. Therefore, the virus and the detection primer designed based on the virus provided by the invention have practical significance and are very important for monitoring the health condition of the cultured tilapia.
Disclosure of Invention
The invention aims to provide a specific gene of Tilapia parvovirus TiPV, the gene is shown in SEQ ID NO.1, a primer designed by utilizing the gene difference of the gene and other viruses can be used for detecting the Tilapia parvovirus TiPV, the preservation number of the Tilapia parvovirus TiPV is CCTCC NO: v201856.
The invention also aims to provide a CPA detection primer designed based on the sequence shown in SEQ ID NO.1, wherein the primer is 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC; 2a: GAACCGGGTCTTGAGTTGT; CGCTGCGGAGAGCTTATCGG a; TGCCACCGATAGTGGACG for 4 s; CGGAATTGCTGGATCCGTG a.
The last purpose of the invention is to provide the application of the primer which is designed by utilizing the polynucleotide shown in SEQ ID NO.1 or based on the polynucleotide shown in SEQ ID NO.1 in preparing the tilapia parvovirus detection kit.
In order to achieve the purpose, the invention adopts the following technical measures:
the specific gene of Tilapia parvovirus TiPV is shown as SEQ ID NO.1, and the preservation number of the Tilapia parvovirus TiPV is CCTCC NO: v201856.
Primers designed based on the gene difference between the gene and other viruses also belong to the protection scope of the invention.
A TiPVCPA detection primer for tilapia parvovirus comprises 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC; 2a: GAACCGGGTCTTGAGTTGT; CGCTGCGGAGAGCTTATCGG a; TGCCACCGATAGTGGACG for 4 s; CGGAATTGCTGGATCCGTG a.
An application of Tilapia parvovirus TiPV CPA detection primer or a sequence shown in SEQ ID NO.1 comprises the step of preparing a detection kit for detecting Tilapia parvovirus by using the primer or the sequence or directly using the detection kit for detecting Tilapia parvovirus.
Compared with the prior art, the invention has the following advantages:
1. the invention separates the parvovirus with pathogenicity from the diseased tilapia and has pioneering significance for the research of the tilapia parvovirus.
2. The method of the invention is rapid, simple and convenient: the CPA method established by the invention only needs hours, and greatly improves the detection efficiency compared with the traditional virus separation method for days.
3. The method of the invention has strong specificity: the recognition of the specific sequence region of the target sequence by the two pairs of primers ensures the high specificity of CPA method amplification.
Detailed Description
The technical scheme of the invention is a conventional scheme in the field if not specifically stated, and the used reagents or raw materials are purchased from commercial sources or published if not specifically stated.
Example 1:
tilapia parvovirus TiPV is isolated as follows:
1) taking out and grinding tissues such as spleen and kidney of tilapia carrying viruses, diluting the tissues by 100 times, centrifuging at 2000rpm, taking supernatant, filtering, inoculating the supernatant to a tilapia kidney cell line (TilapiaKidney, TiK) monolayer, and culturing at 25 ℃ for 7 days to generate cytopathic effect (CPE), wherein TiK cells are seen to shrink and become round, the refractivity is increased, part of cells begin to fall off, and the cell monolayer begins to break.
Culture medium: m199, 10% fetal bovine serum, pH 7.0-7.2
2) Collecting pathological cells, and performing morphological identification by using a transmission electron microscope.
3) Collecting viruses, sequencing and obtaining a specific gene of TiPV of tilapia parvovirus, wherein the gene is shown as SEQ ID NO. 1.
Morphological characteristics of tilapia parvovirus: the virus is spherical, has no envelope and conforms to the shape of a typical parvovirus, and is sent to a China center for type culture Collection for preservation in 2018, 9 and 20 months, and is classified and named as follows: tilapia parvovirus (Tilapia parvovirus, TiPV) TiPV, accession number: CCTCC NO: v201856, address: wuhan university in Wuhan, China.
Example 2:
primers designed based on the difference between the specific gene (shown in SEQ ID NO. 1) of Tilapia parvovirus TiPV and other virus genes are as follows:
a TiPV LAMP detection primer for Tilapia parvovirus comprises 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC; 2a: GAACCGGGTCTTGAGTTGT; CGCTGCGGAGAGCTTATCGG a; TGCCACCGATAGTGGACG for 4 s; CGGAATTGCTGGATCCGTG a.
Example 3:
the method for detecting the tilapia parvovirus by using the tilapia parvovirus TiPV CPA detection primer comprises the following steps:
1. extracting total DNA of tilapia tissues or infected cells: tissue DNA was extracted using omega (USA) DNA extraction kit.
CPA reaction amplification:
a25. mu.l reaction was used: 10 is rich
Reaction Buffer 2.5. mu.L, Betaine 0.7mol/L, Bst DNA polymerase 8U, primer 2a1s 1.0.0. mu. mol/L, primers 2a, 3a each 0.5. mu. mol/L, primers 4s, 5a each 0.6. mu. mol/L, Mg2+8.0mmol/L, dNTPs 1.2mmol/L, template DNA 1. mu.L, and nuclease-free water to 25. mu.L. The optimal reaction condition is isothermal amplification at 63 ℃ for 60 min.
2a1s:GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC；2a:GAACCGGGTCTTGAGTTGT；3a:CGCTGCGGAGAGCTTATCGG；4s:TGCCACCGATAGTGGACG；5a:CGGAATTGCTGGATCCGTG。
Analysis of amplification products of CPA reaction:
1. taking 5 mu L of amplification product, carrying out electrophoresis by using 2% agarose gel, then placing the amplification product in a gel imaging system for imaging, wherein an electrophoresis picture shows a ladder-shaped CPA characteristic band, and the result is positive; if there is no band, the result is negative.
2. And (3) carrying out visual detection on the reaction product by using a nucleic acid detection test strip after the reaction is finished, dripping the product into 5 mu L of the test strip, standing for 1-5 min, and observing the result, wherein if two red lines appear: one is located in the detection area (T), the other is located in the quality control area (C), the detection is judged to be positive, namely, a reaction occurs; if only one red line located in the quality control area appears, judging that the test result is negative; if no red line appears, the detection result is invalid.
Example 4:
tilapia parvovirus CPA detection primer specificity test
The method described in example 3 is used to detect the virus sample to be detected in table 1, and a negative control group (double distilled water) and a positive control group (TiPV) are set at the same time, wherein the virus sample to be detected specifically comprises: giant Salamander Iridovirus Virus (GSIV), carp herpesvirus II (CyHV-2), Koi Herpesvirus (KHV), Carp Edema Virus (CEV) and tilapia lake virus (TiLV).
TABLE 1 Tilapia parvovirus CPA detection kit specificity test
| Test number | Name (R) | Amplification results |
| 1 | TiPV | + |
| 2 | CyHV-2 | － |
| 3 | KHV | － |
| 4 | CEV | － |
| 5 | TiLV | － |
The results are shown in table 1, only the positive control group (TiPV) can detect the ladder-shaped characteristic band, and the rest groups have no characteristic band, which indicates that the TiPVCPA detection primer for the tilapia parvovirus has strong specificity.
Example 5:
sensitivity of tilapia parvovirus CPA detection primer
The primer CPA provided by the invention was used for detection of the detection limit by 10-fold gradient dilution of the TiPVDNA template at a concentration of 2100 ng/. mu.L using the method of example 3, and the results showed that 105The double dilution also detected the band of interest, i.e., the lowest detectable 21.00 pg/. mu.L of DNA template.
Example 6:
the application of Tilapia parvovirus TiPV CPA detection primers in the preparation of Tilapia parvovirus detection kits comprises the following steps:
1. extracting total DNA of tilapia tissues: extracting total DNA of tilapia mossambica spleen and kidney and other viscera tissues infected with TiPV by using an omega (USA) kit, and storing the obtained DNA template at-20 ℃ for later use.
CPA reaction amplification:
a25. mu.l reaction was used: 10 is rich
Reaction Buffer 2.5. mu.L, Betaine 0.7mol/L, Bst DNA polymerase 8U, primer 2a1s 1.0.0. mu. mol/L, primers 2a, 3a each 0.5. mu. mol/L, primers 4s, 5a each 0.6. mu. mol/L,mg2+8.0mmol/L, dNTPs 1.2mmol/L, template DNA 1. mu.L, and nuclease-free water to 25. mu.L. The optimal reaction condition is isothermal amplification at 63 ℃ for 60 min. 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC; 2a: GAACCGGGTCTTGAGTTGT; CGCTGCGGAGAGCTTATCGG a; TGCCACCGATAGTGGACG for 4 s; CGGAATTGCTGGATCCGTG a.
CPA reaction amplification product analysis:
1. taking 5 mu L of amplification product, carrying out electrophoresis by using 2% agarose gel, then placing the amplification product in a gel imaging system for imaging, wherein an electrophoresis picture shows a ladder-shaped CPA characteristic band, and the result is positive; if there is no band, the result is negative.
2. And (3) carrying out visual detection on the reaction product by using a nucleic acid detection test strip after the reaction is finished, dripping the product into 5 mu L of the test strip, standing for 1-5 min, and observing the result, wherein if two red lines appear: one is located in the detection area (T), the other is located in the quality control area (C), the detection is judged to be positive, namely, a reaction occurs; if only one red line located in the quality control area appears, judging that the test result is negative; if no red line appears, the detection result is invalid.
The result shows that the positive control (Tilapia parvovirus TiPV) shows a trapezoidal characteristic band, the negative control (water) has no obvious band, and the Tilapia tissue sample infected with Tilapia parvovirus TiPV from No.1 to No. 5 shows an obvious trapezoidal characteristic band, which indicates that the samples from 1 to 5 are positive to Tilapia parvovirus and are consistent with the actual situation.
Sequence listing
<110> Changjiang aquatic products institute of aquatic science and research in China
<120> TiPV CPA detection primer for tilapia parvovirus and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 212
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgccaccgat agtggacgcg ggagccgaca aattacagtc tttcctggga agcgctgcgg 60
agagcttatc gggggccaca accgagagcc tcccagcaca ggcggcgagc caagtagcaa 120
acgcgccaac acgggaggac ttatcggcag cgcctgcacc acagctgagt acaactcaag 180
acccggttcc gagcacggat ccagcaattc cg 212
<210> 2
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaaccgggtc ttgagttgtg ccgacaaatt acagtc 36
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaaccgggtc ttgagttgt 19
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cgctgcggag agcttatcgg 20
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgccaccgat agtggacg 18
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cggaattgct ggatccgtg 19
Claims (3)
1. A Tilapia parvovirus (Tilapia parvovirus) TiPV specific gene is shown in SEQ ID NO.1, and the preservation number of the Tilapia parvovirus (Tilapia parvovirus) TiPV is as follows: CCTCC NO: v201856.
2. A primer designed based on the specific gene of claim 1, wherein the primer is: 2a1s: GAACCGGGTCTTGAGTTGTGCCGACAAATTACAGTC, 2a GAACCGGGTCTTGAGTTGT, 3a CGCTGCGGAGAGCTTATCGG, 4s TGCCACCGATAGTGGACG, and 5a CGGAATTGCTGGATCCGTG.
3. The application of a reagent for detecting the gene of claim 1 or the primer of claim 2 in preparing a tilapia parvovirus detection kit.
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| CN110042174A (en) * | 2019-04-09 | 2019-07-23 | 广东省农业科学院动物卫生研究所 | The detection method and kit of jewfish irido virus |
| 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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| CN107974515B (en) * | 2017-11-27 | 2021-05-14 | 广州双螺旋基因技术有限公司 | Constant-temperature rapid detection kit for tilapia lake Luo virus |
| CN108220483A (en) * | 2018-02-12 | 2018-06-29 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of reagent, detection method and application for Tilapia mossambica lake viral diagnosis |
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