CN118064644A - Large yellow croaker swelling cell virus typing detection primer, kit and application thereof - Google Patents
Large yellow croaker swelling cell virus typing detection primer, kit and application thereof Download PDFInfo
- Publication number
- CN118064644A CN118064644A CN202410263374.6A CN202410263374A CN118064644A CN 118064644 A CN118064644 A CN 118064644A CN 202410263374 A CN202410263374 A CN 202410263374A CN 118064644 A CN118064644 A CN 118064644A
- Authority
- CN
- China
- Prior art keywords
- yellow croaker
- large yellow
- cell virus
- primer
- pcr
- 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.)
- Pending
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 68
- 241001596950 Larimichthys crocea Species 0.000 title claims abstract description 63
- 238000001514 detection method Methods 0.000 title claims abstract description 55
- 230000008961 swelling Effects 0.000 title claims abstract description 45
- 210000004027 cell Anatomy 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 15
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 12
- 230000003321 amplification Effects 0.000 claims abstract description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 11
- 201000010099 disease Diseases 0.000 claims abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 10
- 238000011841 epidemiological investigation Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 21
- 239000013641 positive control Substances 0.000 claims description 16
- 241000701372 Iridovirus Species 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 150000007523 nucleic acids Chemical group 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000013399 early diagnosis Methods 0.000 abstract description 4
- 241001606461 Large yellow croaker iridovirus Species 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 3
- 238000003205 genotyping method Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 24
- 241001051708 Cyprinid herpesvirus 3 Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 241000049502 Singapore grouper iridovirus Species 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 3
- 241000589516 Pseudomonas Species 0.000 description 3
- 241000404975 Synchiropus splendidus Species 0.000 description 3
- 241000607594 Vibrio alginolyticus Species 0.000 description 3
- 241000269814 Acanthopagrus latus Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000920592 Red seabream iridovirus Species 0.000 description 2
- 241001492212 Striped Jack nervous necrosis virus Species 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 108091006112 ATPases Proteins 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- 241000357439 Epinephelus Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000593508 Garcinia Species 0.000 description 1
- 235000000885 Garcinia xanthochymus Nutrition 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 241001417534 Lutjanidae Species 0.000 description 1
- 241000966621 Megalocytivirus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001222097 Xenocypris argentea Species 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000008642 heat stress Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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
- C12Q1/686—Polymerase chain reaction [PCR]
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)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a large yellow croaker swelling cell virus typing detection primer, a kit and application thereof. The invention designs a primer for detecting the large yellow croaker swelling cell virus, and based on the primer, the large yellow croaker swelling cell virus is detected by a specific method amplification system and conditions. The PCR detection primer has strong specificity and high sensitivity, and can identify whether the large yellow croaker tissue contains the tumor cell virus or not. The invention can be used for detecting the typing of the large yellow croaker swelling cell virus, provides early diagnosis and genotyping for the large yellow croaker iridovirus disease, and provides effective technical support for molecular epidemiological investigation of the large yellow croaker iridovirus disease.
Description
Technical Field
The invention relates to the technical field of aquatic disease detection and identification, in particular to a primer and a kit for detecting the type of a tumor cell virus and application thereof.
Background
Iridovirus disease is one of the main diseases in the current seedling stage of large yellow croaker, and the death rate of the fish fry with disease is 40-80%, so that huge economic loss can be caused. The pathogen is an iridovirus of the genus megaly cell iridovirus (Megalocytivirus). The scholars at home and abroad carry out genetic diversity analysis on the swelling cell viruses through the mcp and ATPase sequences, and the swelling cell viruses can be divided into at least 3 genotypes. Through whole genome analysis and mcp sequence analysis of different isolates of large yellow croaker swelling cell viruses, 2 subtypes of large yellow croaker swelling cell viruses exist, namely FD201807 (LMIV) type and SA type. However, no method for rapidly detecting the large yellow croaker swelling cell virus in a typing manner is currently known. Therefore, the invention provides a primer and a kit for detecting the tumor cell virus genotyping so as to solve the problems.
Disclosure of Invention
(1) Technical problem to be solved
The invention solves the technical problems that the large yellow croaker swelling cell virus typing detection cannot be rapidly performed in the prior art, and early diagnosis analysis and disease prevention cannot be provided, and therefore provides a swelling cell virus typing detection primer, a kit and application thereof.
In order to solve the technical problems, the invention aims to provide a large yellow croaker swelling cell virus typing detection primer;
The invention further aims at providing a kit for large yellow croaker swelling cell virus typing detection;
The invention further aims at providing a PCR detection method for large yellow croaker swelling cell virus typing detection;
The invention also aims to provide application of the primer in preparation of a large yellow croaker swelling cell virus typing detection reagent.
The invention can be used for high-sensitivity and specific molecular detection of large yellow croaker swelling cell viruses, and can also be used for early diagnosis of large yellow croaker iridovirus diseases, virus typing detection and molecular epidemiological investigation.
(2) Technical proposal
In order to solve the above technical problem, a first aspect of the present invention provides:
A large yellow croaker swelling cell virus typing detection primer is 852F and 1272R, and the nucleotide sequences of the primers are respectively as follows:
852F：5’-TATACTGGAGGACGACGAT-3’，
1272R：5’-GCACTCTTCGCATGCGTTATC-3’。
In a second aspect of the invention, there is provided:
A kit for large yellow croaker swelling cell virus typing detection comprises the primer, PCR reaction premix and positive control.
The above-mentioned PCR reaction premix includes reagents conventionally used in the art, such as: taq DNA polymerase, dNTPs, mg 2+, taq enzyme buffer.
Further, the kit further comprises a positive control; the positive control is a recombinant plasmid containing ORF026 nucleic acid sequence of large yellow croaker swelling cell virus FD201807 strain.
In a third aspect of the invention, there is provided:
a PCR detection method for large yellow croaker swelling cell virus typing comprises the following steps:
1) Extracting DNA of a large yellow croaker sample or a virus liquid sample for standby;
2) Performing PCR amplification by using DNA of a large yellow croaker sample or a virus liquid sample as a template and using the primers 852F and 1272R or the kit described above to obtain an amplification product A;
3) And (3) taking the PCR amplification product A to carry out 1% agarose gel electrophoresis detection, comparing with a positive control, judging whether the sample has FD201807 (LMIV) tumor cell virus, and if the sample is amplified to have a band with the size of about 421bp, determining the sample as positive.
The step 1) of extracting DNA from the sample to be tested includes extracting DNA from the sample by using DNA extraction technology conventional in the art or using existing DNA extraction kit.
Further, the PCR amplification system in the step 2) is as follows: 10. Mu.L of the premix solution, 1. Mu.L of the template DNA, 1. Mu.L of each of the primer 852F and the primer 1272R at a concentration of 10. Mu. Mol/L, and 7. Mu.L of sterilized water.
Further, the amplification procedure of the PCR amplification in step 2) is: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 35s, amplification for 30 cycles; finally, the extension is carried out for 5min at 72 ℃.
In a fourth aspect of the invention, there is provided:
the application of the primer in preparing a detection reagent for large yellow croaker swelling cell virus typing.
In a fifth aspect of the invention, there is provided:
The PCR detection method is applied to prevention of iridovirus disease and epidemiological investigation in large yellow croaker culture, and is especially iridovirus of the genus iridovirus of the tumor cells.
(3) Advantageous effects
The beneficial effects of the invention are as follows: the invention designs 1 pair of primers aiming at the ORF026 specific region of the FD201807-LMIV type large yellow croaker swelling cell virus, provides a PCR detection method for large yellow croaker swelling cell virus typing, solves the problem that the prior art lacks a rapid detection method for large yellow croaker swelling cell virus typing, has high detection rate, simple and easy operation, short time consumption, low detection cost and strong specificity, is suitable for rapid diagnosis, can realize specific and sensitive early diagnosis of the FD201807-LMIV type large yellow croaker swelling cell virus, and has stronger reliability.
Drawings
The present invention will be further described with reference to the drawings and examples.
FIG. 1 is a gel electrophoresis chart of the amplification of PCR primers or kits described in example 1 of the present invention: wherein M is DNA molecular mass standard DL2000, lane 1 is positive control, lane 2 is negative control;
FIG. 2 shows gel electrophoresis obtained by performing a specificity test on the PCR primer or the kit described in example 2 of the present invention, wherein M is a DNA molecular mass standard DL2000, lane 1 is a positive control, lane 2 is a healthy fish large yellow croaker tissue, lane 3 is a red sea bream iridovirus (RSIV), lane 4 is a Epinephelus singapore iridovirus (SGIV), lane 5 is a yellow fin sea bream ascites iridovirus (SBV), lane 6 is a mandarin fish frog virus (MRV), lane 7 is a Koi Herpesvirus (KHV), lane 8 is a Nerve Necrosis Virus (NNV), lane 9 is a Vibrio alginolyticus, and lane 10 is Pseudomonas proteus.
FIG. 3 is a gel electrophoresis chart obtained by performing a sensitivity test on the PCR primer or the kit described in the embodiment 3 of the present invention, wherein M is a DNA molecular mass standard DL2000, lanes 1 to 8 are 107、106、105、104、103、102、101、100copies/μL;, and lane 9 is a negative control, respectively.
FIG. 4 is an electrophoresis chart of the PCR primer or the kit for typing of large yellow croaker tumor cell virus in the sample described in the example 4 of the present invention, wherein M is a DNA molecular mass standard DL2000, lane 0 is a negative control, lane 20 is a positive control, and lanes 1 to 19 correspond to the detected large yellow croaker tissue samples 1 to 19, respectively.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.
The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.
Example 1 preparation of a large yellow croaker swelling cell virus typing detection kit:
1. primer design and Synthesis
The large yellow croaker swelling cell virus typing detection primer is synthesized by professional companies according to registered large yellow croaker swelling cell virus genome sequences (OQ 475017.1, AY779031.1, OL774655.1, OL774654.1 and OL 774653.1) in GenBank, various biological software are comprehensively applied, ORF026 specific regions for genotyping identification are screened out through comparison analysis, typing primer 852F and primer 1272R are skillfully designed according to the regions, the primers are synthesized into freeze-dried powder, and the freeze-dried powder is diluted to the working concentration of 10 mu M by sterile water.
The nucleotide sequences of primer pair 852F, 1272R are as follows:
852F：5’-TATACTGGAGGACGACGAT-3’，
1272R：5’-GCACTCTTCGCATGCGTTATC-3’。
2. PCR reaction premix
The PCR reaction premix used in the invention contains Taq enzyme, dNTP, mg 2+, buffer solution and other components required by conventional PCR.
3. Positive control
A positive PCR product with the size of 421bp amplified by DNA of a large yellow croaker swelling cell virus FD201807 strain is connected to a 19-T carrier, heat stress is converted into escherichia coli DH5 alpha, positive cloning bacteria are screened, positive recombinant plasmids are extracted after the positive cloning bacteria are cultured overnight by LB broth containing 100 mug/mL ampicillin, and the positive recombinant plasmids are confirmed after PCR detection and sequencing, wherein the plasmid DNA is a positive control used by the kit, and the sequence is shown as SEQ ID NO 3.
4. The primer, the PCR reaction premix and the positive control form the large yellow croaker swelling cell virus typing PCR detection kit.
Example 2 specific assay
To determine the accuracy of the detection method, a detection-specific test is developed. The cDNA library of positive plasmid, red sea bream iridovirus (RSIV), singapore Grouper Iridovirus (SGIV), mandarin fish frog virus (MRV), yellow fin sea bream ascites iridovirus (SBV), koi Herpesvirus (KHV), pseudomonas deformans, vibrio alginolyticus, genomic DNA of healthy large yellow croaker spleen tissue and Nervous Necrosis Virus (NNV) genome are used as PCR amplification templates, and primer pairs 852F and 1272R are used for amplification.
The total volume of the PCR reaction is 20 mu L, and the optimal reaction system is as follows: 10. Mu.L of PCR premix, 1. Mu.L of template DNA, 1. Mu.L of primer 852F and 1. Mu.L of primer 1272R each at a concentration of 10. Mu. Mol/L, and 7. Mu.L of sterilized water.
The PCR amplification procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 35s, amplification for 30 cycles; finally, the extension is carried out for 5min at 72 ℃.
After the reaction, 6 mu LPCR products were taken for gel electrophoresis, and specific PCR detection was observed.
The primer and the method of the invention are adopted, a positive control sample is used for amplifying a strip with the size of about 421bp, and cDNA libraries of the genome DNA of Red Snapper Iridovirus (RSIV), sinorhizoma Garcinia Iridovirus (SGIV), mandarin fish frog virus (MRV), yellow fin snapper ascites iridovirus (SBV), koi Herpesvirus (KHV), pseudomonas deformans, vibrio alginolyticus, healthy large yellow croaker spleen tissues and Nervous Necrosis Virus (NNV) are used as PCR template amplification products, so that the primer and the method have good specificity (see in figure 2).
Example 3 sensitivity detection
Extracting positive plasmid genome, measuring the concentration of positive plasmid by using Nanodrop2000, respectively diluting to 10 6、105、104、103、102、101、100 copies/. Mu.L, and amplifying according to PCR method of large yellow croaker swelling cell virus typing detection as template. As shown in FIG. 3, the detection result of the sensitivity shows that the template is 10 6、105、104、103、102 copies/. Mu.L positive plasmid, and the positive plasmids have positive bands with the size of about 421bp, so that the detection method can detect 10 2 copies at the lowest and has high sensitivity.
Example 4 detection of large yellow croaker tumor cell Virus typing Using the primers or kits designed in example 1
1. Preparation of detection sample and template DNA
Samples 1-18 are large yellow croakers collected from Ningde cultivation areas in 2018-2022, and samples 1-18 are positive in PCR detection by adopting 1F/R primers in national standard GB/T36191-2018 and negative in detection by adopting the same method as sample 19. Clone sequencing analysis of the whole segment of gene of the virus mcp of the positive sample for detecting the tumor cell virus determines the virus typing as shown in table 1. And 5 fish are taken from each batch of samples, 0.1-0.2 g of each fish is taken, mixed and ground into tissue homogenate, and DNA of a sample to be detected is prepared according to the conventional commercial animal tissue genome DNA extraction kit step and is used as DNA of a large yellow croaker swelling cell virus typing detection template.
TABLE 1 information on sample of Large yellow croaker for typing detection
2. PCR amplification
Taking the DNA of the sample to be detected prepared in the step 1 as a template of 1 mu L, and detecting according to a PCR method for large yellow croaker swelling cell virus typing detection.
3. Agarose gel electrophoresis
And (3) taking PCR amplification products to carry out 1% agarose gel electrophoresis detection, and carrying out 120V constant-pressure electrophoresis for 40min. The results were observed by a gel imaging system or blue light meter. Under normal conditions of detection, the positive control should have 1 band of 421bp (lane 20 in FIG. 4) and the negative control should have no corresponding band (lane 0 in FIG. 4). If the comparison result does not accord with the above, the detection is invalid and the detection needs to be repeated. If the comparison result is normal, judging positive if the sample to be detected detects a band of about 421 bp; and judging the sample to be tested as negative if no corresponding strip exists.
4. Experimental results
The results of the tests performed on samples 1-17 are shown in FIG. 4 (see also Table 1 above), where the positive control showed a band of about 421bp in size, the negative control had no corresponding band, and samples 2, 4, 17, 18 showed a band of about 421bp in size. Sample 2, sample 4, sample 17 and sample 18 are judged to be positive for FD type and are consistent with the typing results of the whole-segment gene sequences of the virus mcp in each sample respectively, which proves that the method or the kit can successfully carry out typing detection on the large yellow croaker swelling cell virus and can provide guarantee for the effectiveness and the scale usability of the kit.
As can be seen from FIG. 4, the PCR detection primer pair of the application has the specificity identification degree on the large yellow croaker swelling cell viruses, can distinguish the large yellow croaker swelling cell viruses from other microorganism bacteria, has high specificity and strong identification degree, can be used for detecting the swelling cell viruses in the large yellow croaker culture environment, has important guiding significance on disease prevention and control in the large yellow croaker culture, can detect whether the large yellow croaker swelling cell viruses are contained in the culture environment without occurrence of the diseases, has the advanced prevention effect on the non-occurrence of the large yellow croaker, and has strong practicability.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all changes and modifications that come within the spirit of the invention are desired to be protected.
Claims (8)
1. The large yellow croaker swelling cell virus typing detection primer is characterized in that the primers are 852F and 1272R, and the nucleotide sequences of the primers are respectively as follows:
852F：5’-TATACTGGAGGACGACGAT-3’，
1272R：5’-GCACTCTTCGCATGCGTTATC-3’。
2. a large yellow croaker swelling cell virus typing detection kit is characterized by comprising the primer pair, PCR reaction premix and positive control.
3. The kit according to claim 2, wherein the positive control is a recombinant plasmid containing the nucleic acid sequence of ORF026 of the yellow croaker's tumor cell virus FD201807 strain.
4. A PCR method for large yellow croaker swelling cell virus typing detection is characterized in that: the method comprises the following steps:
1) Extracting DNA of a large yellow croaker sample or a virus liquid sample for standby;
2) Performing PCR amplification by using DNA of a large yellow croaker sample or a virus liquid sample as a template and using primers 852F and 1272R described in claim 1 or a kit described in claim 2 to obtain an amplification product A;
3) And (3) taking the PCR amplification product A to carry out 1% agarose gel electrophoresis detection, comparing with a positive control, judging whether the sample has FD201807-LMIV type tumor cell virus, and if the sample is amplified to have a band with the size of about 421bp, determining that the sample is positive.
5. The PCR method for detecting the large yellow croaker tumor cell virus typing according to claim 4, wherein:
The PCR amplification system in the step 2) is as follows: 10. Mu.L of the premix solution, 1. Mu.L of the template DNA, 1. Mu.L of each of the primer 852F and the primer 1272R at a concentration of 10. Mu. Mol/L, and 7. Mu.L of sterilized water.
6. The PCR method for detecting the large yellow croaker tumor cell virus typing according to claim 4, wherein:
the amplification procedure for PCR amplification in step 2) is: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 35s, amplification for 30 cycles; finally, the extension is carried out for 5min at 72 ℃.
7. The use of the large yellow croaker swelling cell virus typing detection primer as claimed in claim 1 in preparation of large yellow croaker swelling cell virus typing detection reagent.
8. The use of the PCR detection method according to claim 4 for the prevention of iridovirus disease and epidemiological investigation in large yellow croaker culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410263374.6A CN118064644A (en) | 2024-03-07 | 2024-03-07 | Large yellow croaker swelling cell virus typing detection primer, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410263374.6A CN118064644A (en) | 2024-03-07 | 2024-03-07 | Large yellow croaker swelling cell virus typing detection primer, kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118064644A true CN118064644A (en) | 2024-05-24 |
Family
ID=91095015
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410263374.6A Pending CN118064644A (en) | 2024-03-07 | 2024-03-07 | Large yellow croaker swelling cell virus typing detection primer, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118064644A (en) |
-
2024
- 2024-03-07 CN CN202410263374.6A patent/CN118064644A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111074000B (en) | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain from double gene deletion strain | |
| CN110777220B (en) | Primer group, probe, RPA test strip kit and identification method | |
| CN112322764B (en) | Detection kit and detection method for brucella | |
| CN110904239B (en) | Detection kit and detection method for lung cancer related molecular marker gene mutation | |
| CN107988326A (en) | Prawn Acute Hepatic pancreatic necrosis（AHPND）RAA constant temperature fluorescence detection method and reagent | |
| CN113136429A (en) | Detection kit and detection method for IDH1 or IDH2 gene mutation | |
| WO2020199441A1 (en) | Use of primer probe combination and kit thereof in hbv detection | |
| CN115058543B (en) | Primer group and kit for identifying Delta variant strain and Omicron variant strain | |
| CN111172303A (en) | Mycobacterium tuberculosis drug resistance detection kit and mycobacterium tuberculosis drug resistance detection method | |
| CN110616272A (en) | Primer group for detecting prawn enterocytozoon hepatica and kit containing primer group | |
| CN113637778A (en) | Kit and method for detecting brucella | |
| CN114410844A (en) | Method for determining RCR negative and positive in CAR-T cell product by detecting GALV and ALB copy number through multiple qPCR (quantitative polymerase chain reaction) | |
| CN110938711A (en) | Real-time fluorescent RAA primer, probe and kit for detecting avian infectious laryngotracheitis virus and using method of real-time fluorescent RAA primer, probe and kit | |
| CN110373454A (en) | A kind of kit and method of joint-detection EGFR genetic mutation | |
| CN114015792A (en) | Fluorescent kit for detecting brucella and detection method | |
| CN107190103B (en) | Multiplex PCR primer group, kit and method for simultaneously detecting three fish viruses | |
| CN109811073B (en) | Primer for rapidly detecting streptococcus agalactiae and streptococcus iniae at early stage by double PCR (polymerase chain reaction) and application of primer | |
| CN111363842A (en) | Sequence, kit, method and application for rapidly detecting aspergillus fumigatus | |
| CN109136367B (en) | Method for improving diagnosis efficiency of BRAF gene V600E mutation | |
| CN116042874A (en) | Primer probe combination and kit for detecting multi-drug resistance genes of mycobacterium tuberculosis | |
| CN116042879A (en) | Kit and detection method for detecting brucella wild strain and vaccine strain | |
| CN118064644A (en) | Large yellow croaker swelling cell virus typing detection primer, kit and application thereof | |
| CN116377036A (en) | CRISPR/Cas12 a-based lung cancer patient peripheral blood EGFR gene mutation detection system and method | |
| CN109355362B (en) | High-sensitivity SNPs detection system and application | |
| CN115181803A (en) | Taqman probe qPCR detection primer group for detecting chaulmoogra and application |
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 |