CN109457016B - Quantitative method for spring viremia of carp virus - Google Patents

Abstract

The invention amplifies the nucleoprotein gene N of SVCV by molecular biology technology and constructs recombinant plasmid pMD^TM18-T-SVCV-N, and establishing an absolute quantitative SVCV copy number by taking the plasmid as a template and taking a standard curve based on a TaqMan probe method qPCR. Meanwhile, the Bradford method is used for measuring the protein concentration of the SVCV, the correlation with the copy number of the SVCV measured based on the qPCR method is established, and the method for directly measuring the protein concentration to determine the copy number of the SVCV is established for the first time. The method can realize simple and low-cost determination of the copy number of the SVCV, has important reference significance for the quantification of other viruses, and has important value in relevant research of the SVCV and other viruses.

Description

Quantitative method for spring viremia of carp virus

Technical Field

The invention relates to the technical field of biology, in particular to a virus quantification method.

Background

Spring Viremia of Carp (SVC) is a highly contagious epidemic in fishes of the family Cyprinidae, the etiology of which is the Spring Viremia of Carp Virus (SVCV). SVCV causes the death rate of fish to be more than 90 percent, and poses great threat to the healthy culture of freshwater fish, and is classified as a type of animal epidemic disease in the records of types I, II and III animal epidemic diseases issued by the ministry of agriculture (department of agriculture of the people's republic of China, No. 1125). Moreover, world animal health organization also lists it in the aquatic animal epidemic list (OIE, 2009, chaptor 2.3.8). As for its infectivity, it can be called "avian influenza in aquatic animals" (this disease is parallel to avian influenza and foot-and-mouth disease in our country).

SVC has a wide prevalence of countries, from Europe, the middle east and Russia, and has been spreading in recent years to America (Goodwin & Liopo, 2002; Dikkeboom et al, 2004) and Asia. The United kingdom environmental, Fisheries and Aquaculture Science research center (The Centre for environmental, Fisheries and Aquaculture Science, CEFAS) isolated viruses from goldfish and brocade carp from Beijing, China and identified as SVCV (Goodwin AE, Lipo G. Spring Vision of Carp Vision (SVCV): global station of outleak, Diagnosis, subvariant, and research. The Israeli J ournal of Aquaculture Bamedium, 2009,61(3): 180. sup. 187; Dikkeboal, Radi C, Tooheykurt K, 2004. first of slope vision of carp vision in cement, European design J. 169. The family of food Science, calcium of food J. and Health, emission J. The family of food Science, Inc. 178. The inventor et.7, 2004,24(4): 194-202; stone DM, Ahne W, Denham KL, et al, nucleotide sequence analysis of the viral gene of card virus and pick of viral gene of nucleic acid in the same way as in the previous paragraph, 2003,53:203-

SVCV has a wide host range, can infect cyprinid, cyprinid and non-cyprinid such as silurus asotus, wherein cyprinid is the main and most susceptible host, and SVC has a mortality rate of 70% in 1 year when the disease is outbreak in spring. Cyprinus carpioides is the main breeding species of the aquaculture industry in China, and if SVC is developed, the economic loss effect is unreasonable. [ Paragua, Liu prince, Huang (20514), Chua vitality (SVCV) research progress, Chinese Aquaculture, 2006,13(2): 328) 334]

Although SVC is not a disease of both human and livestock, SVC seriously threatens the healthy development of aquaculture industry, and in view of the mission of economic development of fishery and the responsibility of international trade in China, the work of comprehensively and deeply researching the infection mechanism and prevention and treatment method of SVCV is urgent. For virus-related studies, quantification of viruses is a fundamental but extremely important task. Commonly used methods for virus quantification include: (1) counting the negative infected viral particles by Electron Microscope (EM); (2) the plaque formation test quantification method comprises covering virus-infected monolayer cells with nutrient agar, and counting the plaque formation number on a plate to calculate the number of live viruses in the sample; (3) detecting the virus copy number by real-time fluorescent quantitative PCR (qPCR); (4) microdroplet digital PCR (ddPCR) detects the virus copy number.

Comparative analysis of the above 4 methods:

(I) Electron microscopy

Electron microscopy has a great role in the human recognition of viruses and viral infections, although sometimes considered laborious, outdated or unnecessary, due to its short detection cycle (negative staining) and the possibility of discriminating unknown pathogen assignments, it is suitable for the first line of new-onset pathogen monitoring, since morphology is an important basis for virus classification, and electron microscopy confers ultrastructural features on viruses and visualization of morphogenesis, information which is invaluable for clinical diagnosis of unknown pathogens. However, there are some obvious disadvantages to using electron microscopy for virus quantification, such as:

(1) a support film with good hydrophilicity is used, so that the structure and the dyeing effect of a sample are balanced and consistent, the accuracy of quantitative analysis is ensured, a common copper net cannot meet the sample preparation requirement, and the newly prepared carbon-Formvar (substitute of Formvar or Collodion, Butvar, Pioloform and the like) copper net support film can ensure the uniform distribution of adsorbed particles, but the effect is obviously reduced along with the prolonging of the storage time;

(2) in the negative staining sample process, the degree of affinity between the virus particles and the support membrane or dye will affect the number of adsorbed virus particles, the overall appearance of the virus structure and the change of osmotic pressure, thus affecting quantification;

(3) the equipment requirement is high;

(4) the sample preparation operation is complex, and the requirement on the operation level of an experimenter is high.

(II) plaque assay

The quantitative method for spot test, i.e. spot formation test, is characterized by that the virus liquor which is properly diluted is quantitatively inoculated on the sensitive monolayer cell, after a period of cultivation, a layer of non-solidified agar is covered on the cell, after it is fixed, the culture is continued, the infected monolayer cell is fallen off due to virus proliferation to form macroscopic spot, if in ideal state, one spot is formed by proliferation of one infectious virus particle, and the number of live virus in the sample can be calculated by counting the number of spots. Usually expressed as Plaque Forming Units (PFU) per ml of virus fluid, i.e. PFU/ml. Plaque assay is a commonly used classical method for determining viral titer and isolating viruses, but this method also has significant disadvantages, such as:

(1) the basis of the quantitative method of the plaque formation test is that one infectious particle can form one plaque, but there is a possibility that a plurality of infectious particles simultaneously form one plaque, thereby affecting the accuracy of the quantification. Researchers mix 10 gene-labeled polioviruses (polioviruses) and then perform a plaque experiment, wherein 6 plaques comprise more than one virus in the 123 plaques, and the researches also find that low pH can cause aggregation of virus particles, so that the probability of co-infection is increased;

(2) the plaque formation assay is a quantitative method which takes a long time, and the whole process takes 3 days or more from the preparation of cells, virus inoculation, and the appearance of plaques.

(III) real-time fluorescent quantitative PCR (qPCR) detection

The qPCR has the advantages of high specificity, high sensitivity, capability of quantifying, capability of effectively solving the PCR pollution problem, high automation degree and the like, and is widely applied to the fields of medicine and quarantine of microorganism and animal and plant diseases. The qPCR method can reflect the number of viruses more accurately by determining the copy number of the viral genome. qPCR is a method of measuring the total amount of product after each Polymerase Chain Reaction (PCR) cycle in a DNA amplification reaction using fluorescent chemicals. The qPCR is to detect the PCR process in real time through a fluorescent signal in the PCR amplification process. In the exponential phase of PCR amplification, the Ct value of the template and the initial copy number of the template have a linear relation, so that the Ct value becomes a quantitative basis, and finally, the unknown template is subjected to quantitative analysis through a standard curve. The detection method comprises the following steps: (1) SYBR Green I method: adding excessive SYBR fluorescent dye into a PCR reaction system, wherein the SYBR fluorescent dye emits a fluorescent signal after being specifically doped into a DNA double strand, and SYBR dye molecules which are not doped into the strand do not emit any fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous; (2) TaqMan probe method: the TaqMan fluorescent probe is an oligonucleotide probe, a fluorescent group is connected to the 5 'end of the probe, a quencher is arranged at the 3' end, a pair of primers is added during PCR amplification, a fluorescent probe with the specificity of a target gene sequence is added at the same time, when the probe is complete, a fluorescent signal emitted by a reporter group is absorbed by the quencher, during PCR amplification, the specific probe is combined with a PCR product in an amplification annealing link, the probe is degraded by enzyme digestion through the 5'-3' exonuclease activity of Taq enzyme, so that the reporter fluorescent group and the quencher fluorescent group are separated, a fluorescent monitoring system can receive the fluorescent signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescent signal and the formation of the PCR product are completely synchronous.

Compared with the SYBR Green I method qPCR, the TaqMan probe method qPCR detection gene has the advantages of stronger specificity, higher sensitivity, good repeatability, wide linear range and the like, and is widely applied to multiple fields of biomedicine, agriculture and the like. Chinese scholars Yue and the like establish a method for quantitatively detecting SVCV by qPCR based on a TaqMan probe method. [ Yue Z, et al, development of a sensitive and qualitative assessment for spring vision of car vision based on real-time RT-PCR. J view methods.2008,152(1-2):43-38 ]

(IV) microdroplet digital PCR (ddPCR)

ddPCR is a new generation of PCR technology developed in recent years, and the principle of ddPCR is that before reaction, a reaction system is subjected to micro-titration treatment to form tens of thousands to hundreds of thousands of water-in-oil liquefied micro-droplets, nucleic acid molecules are distributed in each micro-droplet, after PCR amplification, detection is carried out by a micro-droplet detector, and the initial copy number of nucleic acid can be calculated according to the Poisson distribution principle and the number of positive micro-droplets.

Compared with real-time fluorescent quantitative PCR, the method is also based on the TaqMan probe method qPCR, but theoretically, the method has higher sensitivity because the reaction system is subjected to micro-titration. However, ddPCR has some disadvantages, such as expensive equipment and higher detection cost of each part than qPCR; the slow droplet reading speed leads to a detection time 2-3 times that of qPCR; few fluorescence channels (currently only two channels, FAM and VIC); the sample needs to be diluted to a certain concentration for detection; at present, no mature ddPCR commercial kit is available.

Wu and et al explore and establish a method for detecting spring viremia of carp by using digital RT-PCR, and compared with qPCR of the mainstream conventional TaqMan probe method at present, find that the sensitivity of SVCV detected by the two methods is basically consistent, and the detection lower limit is 10^-4And (6) diluting. [ Wubin et al, establishment and application of a method for detecting spring viremia of carp by using digital RT-PCR (reverse transcription-polymerase chain reaction), quarantine of Chinese animals, 2015,32(7) and 72-76.]

In conclusion, the TaqMan probe method qPCR has the advantages of good specificity, high sensitivity, good reproducibility and the like, is a relatively good method for quantitatively detecting viruses including SVCV, but has higher requirements on experimental technology and equipment due to higher cost.

Disclosure of Invention

The invention aims to establish a correlation between a fluorescence quantitative RT-PCR method and a Virus protein quantitative determination method according to the defects of the prior art, realize the quantification of Spring Viremia of Carp Virus (SVCV) by directly determining the content of Virus protein, has accurate method, is used for detecting the purified SVCV, has simple and convenient operation and low cost, and brings great convenience for the related research of the SVCV.

According to one aspect of the invention, SVCV virus particles with high purity are obtained by expanding and culturing SVCV cells (Epithelioma papulosum cytoprini, EPC), repeatedly freezing and thawing the cells to collect SVCV, and then adopting a method combining ultracentrifugation and density gradient centrifugation.

Another aspect of the present invention is to amplify the SVCV gene by molecular biology techniques and clone the SVCV nucleoprotein N gene sequence gene into plasmid pMD^TM18-T, construction of recombinant plasmid pMD^TM18-T-SVCV-N. Then, the design synthesis was used for TaqMan probe based qPCRPrimers and corresponding fluorescent probes, plasmid pMD diluted in gradient^TM18-T-SVCV-N is taken as a template, an SVCV copy number absolute quantification method based on TaqMan probe method qPCR is established, a Linear Regression (Linear Regression) equation between the Ct value (y) of the qPCR and the logarithmized template concentration (x, logs (copies/. mu.l)) is that y is-3.3407 x +40.489, and a correlation coefficient R is²Up to 0.9967.

In yet another aspect of the invention, the method comprises determining the concentration of SVCV protein by using a standard curve of Bovine Serum Albumin (BSA) diluted in a gradient determined by the Bradford method, and then setting the linear regression equation between the concentration (y) of SVCV protein diluted in the determined gradient and the absorbance (OD) value (x) to be y 434.98x-163.01, wherein the correlation coefficient R is²Up to 0.9952. Meanwhile, a TaqMan probe method qPCR-based method is adopted to determine the corresponding gradient dilution SVCV copy number, and then the correlation between the SVCV protein concentration and the SVCV copy number is established, wherein the linear regression equation of the SVCV protein concentration and the SVCV copy number is that y is 3.0 multiplied by 10^-6x +9.4967, correlation coefficient R²Up to 0.9965.

Specifically, the method comprises the following steps:

a method of quantifying a spring viraemia of carp virus, the method comprising:

1) amplifying nucleoprotein N gene from spring viremia of carp virus to be detected, constructing recombinant plasmid, and establishing absolute quantitative SVCV virus copy number by taking the plasmid as a template based on a standard curve made by TaqMan probe method qPCR;

2) determining the protein concentration of the SVCV virus to be detected by using a Bradford method, and establishing the correlation with the copy number of the SVCV detected based on a qPCR method;

3) a quantitative method for determining the copy number of the SVCV virus by directly measuring the protein concentration when the virus is re-detected by the association establishment described in step 2).

1. The quantification method according to claim 1, characterized in that the step 1) comprises:

1a) inoculating viruses to be detected to host cells, carrying out amplification culture, extracting and purifying a sample containing SVCV to prepare a sample solution to be detected;

1b) reverse transcription is carried out on the sample solution to be detected obtained in the step 1a) to extract cDNA of virus to be detected, and recombinant plasmid containing coding SVCV nucleoprotein N gene is prepared;

1c) taking the recombinant plasmid obtained in the step 1b) as a standard template, respectively carrying out real-time fluorescent quantitative PCR reaction by using a dye method and a Taqman probe method, and calculating the DNA copy number of the recombinant plasmid;

1d) a standard curve was prepared with the logarithm of the copy number of the plasmid as the abscissa and the Ct value as the ordinate.

The quantitative method of the present invention, wherein the step 2) comprises:

2a) preparing a protein standard curve by taking the bovine serum albumin diluted in a gradient manner as a standard substance;

2b) diluting the virus solution to be detected in a gradient manner, and calculating the protein concentration of the virus solution of each dilution gradient by using the OD value under the 595nm condition by using the Bradford method through the protein standard curve in the step 2 a);

2c) performing real-time fluorescence quantitative PCR reaction by taking diluted virus liquid as a template, performing reverse transcription on the virus into cDNA, and taking the reversed cDNA as the template;

2d) calculating the copy number of the virus liquid according to the standard curve made in the step 1 d);

2f) correlating the protein concentration measured in step 2b) with the viral copy number measured in step 2 d).

The quantitative method of the present invention, wherein the step 3) comprises:

3a) amplifying and purifying the SVCV virus to be detected again;

3b) performing gradient dilution on the repurified virus solution, and determining the protein concentration of the virus solution of each dilution gradient by using a Bradford method;

3c) calculating the number of copies of the virus from the protein concentration determined in step 3b) on the basis of the correlation established in step 2 f).

More preferably, the quantitative method of the present invention comprises the following steps in sequence:

i) culturing EPC cells in an M199 culture medium, carrying out passage, inoculating viruses to be detected, continuously culturing until 90% of cells have cytopathic effect, and collecting cell and virus mixed liquor;

ii) repeatedly freezing and thawing the collected cell and virus mixed solution, and filtering to remove cell debris to obtain virus primary extract;

iii) centrifugally precipitating the virus primary extract, and re-suspending the virus sample by using a buffer solution to obtain a super-separation virus sample;

iv) preparing sucrose solutions with density gradients of 15%, 30%, 45% and 60%, adding the ultra-separated virus samples on the sucrose solutions with density gradients, centrifuging, and collecting virus distribution areas with liquid in 45% and 30% sucrose layers to prepare purified virus samples;

v) Using SEQ ID No.2 and SEQ ID No.3 as primers, purified viral samples as RNA templates, and Takara reverse transcription kit PrimeScript^TMRT-PCR Kit carries out RT-PCR to amplify cDNA of the nucleoprotein N gene;

vi) constructing recombinant plasmid pMD from the obtained cDNA^TM18-T-SVCV-N and transformed to DH5 α;

vii) design of pMD^TMqPCR primer and Taqman probe of 18-T-SVCV-N, extracting plasmid pMD from bacterial liquid^TM18-T-SVCV-N is used as a standard template, and a dye method and a Taqman probe method are respectively used for carrying out real-time fluorescent quantitative PCR reaction;

the DNA copy number of the recombinant plasmid was calculated according to the following method:

molecular copy (. mu.L) ═ 6.02X 10²³) X (plasmid concentration X10)^-9) V (plasmid bp number × 660)

Taking the logarithm of the copy number of the plasmid as an abscissa and the Ct value as an ordinate to make a standard curve;

viii) diluting a series of concentration gradients by using BSA as a standard substance, then adding a Bradford reagent, measuring absorbance at 595nm by using an enzyme-labeling instrument after reaction, and taking the OD value of the absorbance as a horizontal coordinate and the protein concentration as a vertical coordinate to make a standard curve;

ix) diluting the purified SVCV virus solution in gradient, and then calculating the protein concentration of the virus solution of each dilution gradient by using the absorbance value under 595nm by using a Bradford method through the protein standard curve in the step viii);

x) taking diluted SVCV virus liquid as a template, carrying out reverse transcription on the virus to form cDNA, taking the reversed cDNA as the template, carrying out real-time fluorescence quantitative PCR reaction, and calculating the copy number of the virus liquid according to the standard curve made in the step vii);

performing correlation analysis on the protein concentration of SVCV measured by the Bradford method in the step ix) and the virus copy number measured by the RT-qPCR in the step x) to obtain a regression equation between the protein concentration and the copy number of the gradient dilution;

for the same batch of virus, the copy number of SVCV was calculated by regression equations directly in subsequent studies by determining the protein content of the purified SVCV.

On the basis of a TaqMan probe method qPCR, the method establishes a correlation between a conventional protein quantitative method and TaqMan probe method qPCR molecular detection, establishes an innovative method for determining SVCV copy number directly by Bradford method protein quantification, is used for detecting purified SVCV once established, and has the advantages of simple and convenient operation and low cost.

Drawings

FIG. 1 is an agarose gel electrophoresis chart showing the results of RT-PCR amplification of SVCV-N gene;

in the figure: m: DNA Marker; 1. 2: an SVCV virus stock solution RT-PCR product; 3: negative (PCR product with sterile water instead of template).

FIG. 2 is a nucleic acid electrophoresis chart of PCR products of colonies of the vector to verify whether the target fragment is ligated to the vector after the plasmid transformation;

in the figure: m: DNA Marker; 1-5 different single colonies are taken as templates; 6 negative (PCR product with sterile water instead of template).

FIG. 3 is a graph of sequencing peaks after SVCV-N ligation to a vector;

FIG. 4 is an SDS-PAGE electrophoresis of SVCV protein purified by ultracentrifugation and density gradient centrifugation;

in the figure: 1: ultracentrifugation sedimenting resuspension sample; 2: density gradient centrifugation of the sample layer (uppermost layer) sample; 3: density gradient centrifugation of 15% sucrose layer samples; 4: density gradient centrifugation of 30% sucrose layer samples; 5: density gradient centrifugation of 45% sucrose layer samples; 6: density gradient centrifugation of 60% sucrose layer samples; m protein marker.

FIG. 5 is an electrophoretogram of purified SVCV non-denatured acid collagen;

in the figure: 1: m199 medium containing 10% FBS; 2: SVCV virus stock solution; 3: sucrose density gradient centrifugation of purified SVCV.

FIG. 6 is an electrophoretogram of purified SVCV non-denatured alkaline collagen;

in the figure: 1: m199 medium containing 10% FBS; 2: SVCV virus stock solution; 3: sucrose density gradient centrifugation of purified SVCV.

FIG. 7 shows the Taqman probe method for detecting the amplification curve (A) and the standard curve (A) of the SVCV N gene;

FIG. 8 shows a dissolution curve (A) and a standard curve (A) according to the SYBR dye method;

FIG. 9 protein standard curves by Bradford method;

FIG. 10Taqman probe assay for virus detection and correlation assay of Bradford assay for protein concentration (first batch);

FIG. 11Taqman probe assay detects virus and correlation assay of Bradford assay protein concentration (second batch).

Detailed Description

Test material and apparatus

1. Source of biological experimental material

(1) Coli (e.coli dh5 α) purchased from shanghai mysteries biotechnology limited.

(2) Carp epithelial tumor cells (EPC) and Carp Spring Viremia Virus (Spring Viremia of Carp Virus, SVCV) were provided by Shenzhen entry and exit inspection and quarantine office.

2. Reagent

DL-500DNAmarker and DL-2000DNAmarker were purchased from Takara Bio Inc. ExTaq enzyme and reverse transcription kit PrimeScript^TMRT-PCR Kit and fluorescent quantitative PCR Kit Premix ExTaq^TM(Probe qPCR)、TBGreen^TMPremix Ex Taq^TMII (Tli RNaseH plus), DNA Fragment Purification Kit Mini BEST DNA Fragment Purification Kit Ver.4.0, pMD^TM18-T Vector Cloning Kit was purchased from Takara, Inc., Dada Liang bioengineering (Dada). Medium M199 was purchased from Gbico (USA). Rapid plasmid miniprep kit (D)P105) was purchased from tiangen biochemical technology (beijing) ltd. An improved Bradford protein concentration determination kit, an antibiotic Penicillin-Streptomyces 37.5:1 and the like are purchased from the Biotechnology engineering (Shanghai) GmbH.

Diethylpyrocarbonate (DEPC) treated water, 40% polyacrylamide, Sodium Dodecyl Sulfate (SDS), Tetramethyldiethylamine (TEMED), Ammonium Persulfate (APS), concentrated ampicillin hydrochloride, NaCl, peptone, yeast extract for bacterial culture, agar powder, agarose, methyl green, bromophenol blue, glycerol, KOH, beta-Alanine, Tris, glycine, KCl, Na₂PO₄、KH₂PO₄Serum (FBS), dimethyl sulfoxide (DMSO), HEPES, pancreatin, sodium bicarbonate (NaHCO)₃) Sucrose, etc. are available from bio-engineering (Shanghai) corporation.

3. Instrumentation and equipment

[ example 1 ] amplification and purification of spring viraemia of carp Virus

1.1 amplification of SVCV Virus

Culturing EPC cells in M199 medium containing FBS at 10% under constant temperature of 25 deg.C and 5% CO₂. One day before virus inoculation, EPC cells are subjected to passage, the cell concentration is properly adjusted, the coverage rate of the cells after the cells adhere to the wall of a culture bottle in the next day is about 90%, and the virus is inoculated. Adding virus liquid, slightly shaking the culture bottle to disperse the virus uniformly and contact the cells sufficiently, and then placing the culture bottle in an incubator at 25 ℃ for continuous culture. Cells were observed daily and photographed, and when 90% of the cells had cytopathic effect (CPE), the mixture of cells and virus was collected.

Freezing and thawing the collected cell and virus mixture for 3 times, and filtering with filter membrane with pore diameter of 0.22 μm to remove cell debris to obtain virus primary extractive solution.

1.2 Virus purification

Centrifuging the virus primary extract at 140,000g at 10 deg.C for 3h, discarding the supernatant, and resuspending the virus sample precipitated by ultracentrifugation with appropriate amount of HEPES buffer solution to obtain an ultracentrifugation virus sample.

Preparing 15%, 30%, 45% and 60% sucrose solutions, and carefully adding the 60%, 45%, 30% and 15% sucrose solutions into a centrifuge tube in sequence, wherein the boundary of different sucrose is kept when adding the sucrose solutions, and the sucrose solutions with different concentrations cannot be mixed together. The ultra-viral sample was then carefully added on top of the density gradient sucrose solution and centrifuged at 120,000g for 3h at 10 ℃. After centrifugation, a clear white zone can be seen in the 45% and 30% sucrose layers, which is the distribution zone of SVCV, the liquid in the zone is collected, centrifuged at 140,000g at 10 ℃ for 3h, the supernatant is discarded to remove sucrose, and the supernatant is resuspended in a proper amount of HEPES buffer solution to obtain a purified SVCV virus sample which is used for the next experiment or stored at-80 ℃.

Example 2 acquisition of Gene sequence encoding nucleoprotein N of spring Virus-Hematosis Virus

2.1 acquisition of nucleic acid sequence of nucleoprotein N of spring viremia of carp Virus

Obtaining spring viremia of carp Virus (spring viraemia of carp Virus) on GenBank

The whole genome sequence of SVCV-265 strain, accession number KJ 513477.1. The gene sequence encoding nucleoprotein N was obtained as follows (SEQ ID NO. 1):

ATGAGTGTCATTCGGATCAAAACAAATGCTACAGTTGCTGCCGTGCTTCCGGCTAACGAAGATCAGGCCGATTATCCTTCCACTTTTTTTGAAGGGGGGAATGAGATTAGATTGTATGTTAACAGGGGGGAGAAATTGGATGTTTTAAGGCAATATGTCTATATGGGACTGGTGGAGAAAAACTGTAGGATACAGCATGTGAATGCTTATCTATATGCTGTGCTGAAGGGAGAAAGAGAGCTGCTAGAAGCGGATTGGGATAGCTTTGGGCACAAGATTGGGATTCAGGGGGATAAGATCGGGCCTTTTAACTTGGTGCGAGTGGAAGACATCCCCGACGGGTTACCAGATGGGAAACTGAATGCAGAGGTGAGTGCTGAGGATGATGCATGGCTGCCTCTCTTCTTGCTGGGTCTTTACAGAGTGGGAAGGGCAAGTGAGACTGCATACCGGACTTTGCTGATGGAGTCCCTGATAAAACAGTGTAAGGCAATAAAATCTGACTGGGTGTCTCCTGTAACGGCAACTCACAAATATTTCGATATCTGGGGCAATGATGGGAATTACCTGAAGATTGTGGCCTGTGTGGACATGTTTTACAACCATTTTAAAAAGAGCATTAAAGCAACATTCCGATGGGGAACGATTGTATCACGGTTCAAAGACTGTGCTGCACTCGCCACCCTGGGACATGTTGTCAAAATCACCGGTTTGACCATTGAAGAGGTGTTCACATGGGTACTGCAGACTGAAGTCGCGGATGAGTTAGTCAAAATGATGAAGCCTGGACAGGAGATAGATAAAAGCACGTCTTACATGCCGTACCTGATTGATATGGGAATCTCTGCCAAATCACCATACTCAACAATAAAGAATCCGTCTTTCCATTTCTGGGGGCAGCTTGTTGCTGCATTGTGCCGCTCCAAGAGAGCACTGAACGCAAGACAGCCTGATGAGATTGACTCAATGTCTATCTCAAATGCAAGCTTGCTGATGGCTTACGCATTAGGCAGCAGCCCTGACATTGAGCAGCAATTCAGTACAGGAGACACATACAGAAAACCGCCAAAAGAGGCTTCGTACCTGGTGAGTGAGGAACCGAAAAACCGATCTGTCGTTGAATGGATTGCATGGTATTCTGACGTGGACAACAAACCTACGGATGACATGCTCATGATGGCAAAACGAGTAGCGGGGACTATCTCTGGGCCTCGCGATAATTCAGTTGGCAAATGGATAAAACAAACCTATGGGTAA

2.2 design of primers for amplifying the Nuclear protein N Gene

The sequence was sent to Guangzhou Egyptian Biotechnology Ltd for synthesis using software Primer Premier 5.0 to design a pair of specific primers Fsvcv-N, Rsvcv-N to amplify the nuclear protein N gene. The primer sequences are as follows: Fsvcv-N (5 '-3'): GTGGGAAGGGCAAGTGAGA (SEQ ID NO.2) Rsvcv-N (5 '-3'): TTGTTGTCCACGTCAGAATACC (SEQ ID NO.3)

The target amplification sequence is 731bp, and the sequence is as follows (SEQ ID NO. 4):

GTGGGAAGGGCAAGTGAGACTGCATACCGGACTTTGCTGATGGAGTCCCTGATAAAACAGTGTAAGGCAATAAAATCTGACTGGGTGTCTCCTGTAACGGCAACTCACAAATATTTCGATATCTGGGGCAATGATGGGAATTACCTGAAGATTGTGGCCTGTGTGGACATGTTTTACAACCATTTTAAAAAGAGCATTAAAGCAACATTCCGATGGGGAACGATTGTATCACGGTTCAAAGACTGTGCTGCACTCGCCACCCTGGGACATGTTGTCAAAATCACCGGTTTGACCATTGAAGAGGTGTTCACATGGGTACTGCAGACTGAAGTCGCGGATGAGTTAGTCAAAATGATGAAGCCTGGACAGGAGATAGATAAAAGCACGTCTTACATGCCGTACCTGATTGATATGGGAATCTCTGCCAAATCACCATACTCAACAATAAAGAATCCGTCTTTCCATTTCTGGGGGCAGCTTGTTGCTGCATTGTGCCGCTCCAAGAGAGCACTGAACGCAAGACAGCCTGATGAGATTGACTCAATGTCTATCTCAAATGCAAGCTTGCTGATGGCTTACGCATTAGGCAGCAGCCCTGACATTGAGCAGCAATTCAGTACAGGAGACACATACAGAAAACCGCCAAAAGAGGCTTCGTACCTGGTGAGTGAGGAACCGAAAAACCGATCTGTCGTTGAATGGATTGCATGGTATTCTGACGTGGACAACAA

[ example 3 ] recombinant plasmid pMD^TMConstruction of 18-T-SVCV-N

3.1 obtaining cDNA of Virus SVCV by reverse transcription

Using a stock solution of SVCV virus obtained in the early stage of the laboratory as an RNA template, PrimeScript was prepared using a reverse transcription kit from Takarat^TMRT-PCR Kit RT-PCR was performed.

1) Template RNA denaturation and reverse transcription reaction

The reaction mixture was prepared according to the following table using a clean RNase free PCR tube:

2) the denaturation and annealing reactions were carried out on a PCR apparatus according to the following procedures

65℃5min

4℃

3) The following reverse transcription reaction solution was prepared in the above reaction tube

4) The reverse transcription reaction was performed on a PCR instrument under the following conditions:

30℃10min

42℃30min

95℃5min

4℃

3.2PCR amplification of the target Gene sequence of matrix protein M

1) A PCR reaction solution was prepared with the following composition:

2) the amplification reaction was performed on a PCR instrument according to the Touch down program as follows:

3) the PCR product was electrophoresed on a 2% agarose gel.

3.3 construction of recombinant plasmid pMD^TM18-T-SVCV-N and conversion to DH5 alpha

1) The PCR product obtained above was purified using the Mini BEST DNA Fragment Purification Kit Ver.4.0 Kit of Takara to obtain a purified DNA Fragment.

2) The DNA fragment purified in the above step was used as insert DNA, and a DNA solution was prepared according to the following table, and an equal amount (5. mu.L) of solution I was added thereto, mixed well, and allowed to stand at 16 ℃ for 4 hours and then overnight at 4 ℃.

Sample adding meter

3) Plasmid transformation and sequencing verification

Taking out DH5 alpha competent cells from a refrigerator at minus 80 ℃, placing the cells on ice for slow thawing, taking 5 mu L of reaction liquid after connecting the carriers, adding the reaction liquid into 50 mu L of competent cells, mixing the cells evenly and lightly, standing the cells on ice for 30min, transferring the cells to a constant-temperature metal bath at 42 ℃, thermally stimulating the cells for 45s, thermally stimulating the cells on ice for 5min, adding 250 mu L of LB liquid culture medium into a centrifuge tube, blowing the cells evenly and uniformly, placing the cells in a shaking table, culturing the cells for 1h at 37 ℃ and 200rpm for resuscitation, then sucking 150 mu L of the culture liquid into an LB plate (containing antibiotics ampicillin and Amp) for uniform coating, and reversely culturing the plate in an incubator at 37 ℃ overnight. Picking single colony to 5 mu L of sterile water, taking 1 mu L of the single colony as a template, carrying out PCR amplification by taking FsvcvN and RsvcvN as primers, carrying out agarose gel electrophoresis on an amplification product, taking the rest 4 mu L of the single colony with an expected size fragment amplified in the sterile water for overnight amplification culture, sending the bacterial liquid to Guangzhou Aiji biotechnology Limited company for sequencing the next day to determine whether the sequence and the connection direction of the inserted target gene are correct, and selecting the correct plasmid extracted from the bacterial liquid as a standard product of the next qPCR.

Example 4 real-time fluorescent quantitative PCR calibration Curve

4.1 designing pMD Using software AlleID 6^TMqPCR primer of 18-T-SVCV-N, Taqman probe and primerThe substance and the probe were synthesized by Guangzhou Egyki Biotechnology Ltd. The primer and probe sequences are as follows:

qFsvcvN(5’-3’):AGCTTGTTGCTGCATTGTGC(SEQ ID NO.5)

qRsvcvN(5’-3’):TGCCTAATGCGTAAGCCATCAG(SEQ ID NO.6)

probeN(5’-3’):FAM-CCAAGAGAGCACTGAACGCAAGACAGCC-TAMRA(SEQ ID NO.7)

4.2 extraction of plasmid pMD from bacterial solution Using Rapid plasmid Mini-extraction kit (DP105) from Tiangen Biotechnology Ltd^TM18-T-SVCV-N, diluting the plasmid with 10-fold gradient, using the diluted plasmid as standard template, and using fluorescent quantitative PCR kit Premix ExTaq^TM(Probe qPCR) and TB Green^TMPremix Ex Taq^TMII (Tli RNaseH plus), namely, a real-time fluorescent quantitative PCR reaction is carried out by using a dye method and a Taqman probe method respectively.

The DNA copy number of the recombinant plasmid was calculated according to the following method:

molecular copy (. mu.L) ═ 6.02X 10²³) X (plasmid concentration X10)^-9) V (plasmid bp number × 660)

The standard curve is plotted on the abscissa as the logarithm of the copy number of the plasmid and on the ordinate as the Ct value (the number of cycles that the fluorescence signal in each reaction tube has undergone until reaching a set threshold value, C for cycle and t for threshold). The reaction system of the SYBR dye method has the following reaction parameters of 95 ℃ and 30 s; 95 ℃ for 5 s; 30s at 60 ℃; 95 ℃ for 15 s; 60 ℃ for 1 min; 95 ℃ for 15 s; for a total of 40 cycles, three replicates for each dilution were set:

the reaction system of the Taqman probe method is as follows, the reaction parameter is 95 ℃, 30 s; 95 ℃ for 5 s; 60 ℃, 34s, 40 cycles, three replicates for each dilution:

example 5 purified SVCV was subjected to Native-PAGE

The purity of the purified SVCV was checked by Native-PAGE, and the working solution of the acid Native gel was as follows:

4 Xseparation buffer (0.24M KOH) 0.673g KOH was dissolved in ddH2O, adjusted to pH 4.3, and made up to 50 ml;

4x Stack buffer (0.24M KOH) 0.673KOH was dissolved in ddH2O, adjusted to pH 6.8, made up to 50 ml;

5 × electrophoresis buffer: 17.82g of beta-Alanine was dissolved in dd H2O, adjusted to pH 4.4 and made up to 500 ml.

5x methyl Green protein Loading buffer (10 ml): methyl green 1mg, glycerol 7.5ml, beta-Ala-HCl electrophoresis buffer 2.5 ml.

The working solution of the alkaline non-denatured glue is as follows:

4 × separation gel buffer (1.5M Tris-HCl, pH 8.8): 9.0855g Tris dissolved in ddH2O, adjusted pH to 8.8 with concentrated hydrochloric acid, and made to 50ml volume;

4 × Cascade buffer (0.5M Tris-HCl, pH 6.8): 3.0285g Tris dissolved in ddH2O, adjusted pH to 8.8 with concentrated hydrochloric acid, and made to 50ml volume;

10 Xelectrophoresis buffer (pH8.8Tris-Gly), 15.15g Tris, 72g glycine, water to 500ml, 4 ℃ storage;

5 Xbromophenol blue loading buffer 1M Tris-HCl (pH 6.8)0.6ml, 50% glycerol 5ml, 1% bromophenol blue 1ml, ddH2O 3.4.4 ml; storage at-20 ℃.

The acid and alkaline gels of Native-PAGE are shown in the following table:

Native-PAGE acidic protein gel formula

Native-PAGE (polyacrylamide gel electrophoresis) alkaline protein adhesive formula

10 mul of culture solution and 20 mul of purified SVCV (SVCV) virus stock solution are respectively added with 5x electrophoresis buffer solution, mixed evenly, the gel is pre-electrophoresed for 30min under the constant pressure of 100V without adding any sample, the electrophoresis tank is placed in ice water, electrophoresis is carried out under 100V until the sample enters separation gel, then electrophoresis under the constant pressure of 160V is carried out, the electrophoresis is stopped when methyl green or bromophenol blue moves to the bottommost part of the separation gel, and the electrodes need to be reversed when electrophoresis of acidic non-denatured gel is carried out. Removing concentrated gel, transferring the separated gel into Coomassie brilliant blue staining solution, dyeing for 30min, placing the gel in appropriate amount of decolorizing solution, slowly shaking by horizontal shaking table for 4-6 hr, and changing the decolorizing solution for 3-4 times according to decolorizing condition until the background is clear. And (4) photographing the decolored protein glue by using an ultraviolet imager for storage, observing the result and analyzing.

Example 6 correlation of the concentration of SVCV protein measured by the Bradford method with viral RT-qPCR

1) Standard protein curve by Bradford method

A series of concentration gradients were diluted with BSA in the Bradford kit as standard: 0. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml, 15. mu.g/ml, 20. mu.g/ml, 25. mu.g/ml, 30. mu.g/ml, 100. mu.g/ml, 150. mu.g/ml, 200. mu.g/ml, 250. mu.g/ml, 300. mu.g/ml, three replicates per dilution were set, 20. mu.L was added to a 96-well plate, 200. mu.L of Bradford reagent was then added to each well, and after a reaction time, the absorbance (OD value) was measured at a wavelength of 595nm using a microplate reader, and a standard curve was prepared with the OD value as abscissa and the protein concentration as ordinate.

2) Carrying out double-ratio gradient dilution on purified SVCV virus solution obtained in the early stage of a laboratory, wherein the dilution ratio is 1/2-1/2⁷The total of 7 concentrations were determined by the Bradford method using an OD at 595nm using the protein standard curve of the previous step to determine the protein concentration of the virus solution at each dilution gradient.

3) Using diluted SVCV virus solution as a template and Takara's kit PrimeScript^TMRT-PCR Kit reverses VirusThe cDNA was recorded as cDNA, and the reversed cDNA was used as a template, and Premix ExTaq was used as a kit of Takara^TM(Probe qPCR) and TBGreen^TMPremix Ex Taq^TMII (Tli RNaseH plus) for real-time fluorescent quantitative PCR reaction. The number of copies of the virus fluid was calculated from the standard curve prepared in example 4.

4) The protein concentration of SVCV measured by Bradford method was correlated with the virus copy number measured by RT-qPCR.

Application of the method [ example 7 ]

7.1 reamplification and purification of the viral SVCV according to the method [ example 1 ]

7.2 according to [ example 6 ], the repurified SVCV solutions were diluted in twofold gradient, protein concentration of the virus solution at each dilution gradient was determined by Bradford method, copy number of SVCV was determined at the corresponding gradient based on TaqMan probe method qPCR, and the correlation between SVCV protein concentration and virus copy number of the batch was analyzed.

The results show that:

(1) cloning of SVCV Nuclear protein N Gene sequence Gene to plasmid pMD by amplification of SVCV Gene^TM18-T, construction of recombinant plasmid pMD^TM18-T-SVCV-N, and plasmid sequencing shows that the SVCV nucleoprotein N gene is successfully inserted into pMD^TM18-T vector. The results of gene amplification and plasmid sequencing verification during plasmid construction are shown in fig. 1, fig. 2 and fig. 3, respectively.

(2) SVCV is expanded and cultured by EPC, SVCV is collected by repeatedly freezing and thawing cells, then the SVCV is purified by a method combining ultracentrifugation and density gradient centrifugation, sucrose is removed by ultracentrifugation, the result of SDS-PAGE electrophoresis is shown in figure 4, the result shows that the sample with the highest SVCV purity is located in a 30% sucrose layer of density gradient centrifugation, the SVCV sample in the layer is collected, and then the non-denatured acidic and alkaline collagen electrophoresis detection is carried out, so that the lane in which the purified SVCV sample is located has no protein entering non-denatured albumin, and the high-purity SVCV virus particles are obtained through verification (figure 5, figure 6).

(3) Design and Synthesis of primers and corresponding fluorescent probes for TaqMan-based Probe method qPCR, plasmid pMD diluted in gradient^TM18-T-SVCV-N isThe absolute quantitative method of SVCV copy number based on TaqMan probe method qPCR is established for the template, as can be seen from the amplification curve of figure 7(A), each template gradient has an S-shaped amplification curve, the three stages of exponential amplification period, linear amplification period and platform period of PCR are reflected, and simultaneously, 2 parallel amplification curves of each dilution degree are well matched, which shows that the repeatability of the experiment is good. The method can detect SVCV with 50copies (copies) at the lowest, wherein the SVCV is 50copies to 4.96 multiplied by 10⁹In the copes range, the Ct value (y) of qPCR and the logarithmized template amount (x, Log (copes)) have extremely high Linear correlation, the Linear Regression equation is that y is-3.1996 x +41.543, and the correlation coefficient R²Up to 0.995 (see fig. 7 (B)).

The absolute quantitative method of SVCV copy number by dye method qPCR can be seen from FIG. 8(A), the dissolution curve is a single peak, which indicates that the specificity of the amplification product is good, the method can detect 500copies (copies) of SVCV at the lowest, and the concentration of SVCV is between 500copies to 4.96 multiplied by 10⁹The copies range also has extremely high Linear correlation, and the Linear Regression (Linear Regression) equation is that y is-3.3407 x +40.489, R²Up to 0.997 (see fig. 8 (B)).

Chinese scholars Yue and the like (2008) establish a method for quantitatively detecting SVCV by qPCR based on a TaqMan probe method, and SVCV envelope glycoprotein G is detected. The lowest detectable 40copies (copies) of SVCV at concentrations of 40copies to 4X 10⁷In the copes range, there is a very high Linear correlation between Ct value (y) of qPCR and logarithmized template concentration (x, log (copes)), and the Linear Regression equation is-3.36 x +38.42, R²Is 0.992.

The method for detecting the SVCV nucleoprotein N based on the TaqMan probe method qPCR established by the invention has the advantages of high sensitivity, better linearity and wider linear range.

(3) A Bradford method is used for measuring the Bovine Serum Albumin (BSA) of the gradient dilution to be used as a standard curve, and a linear regression equation between the concentration (y) and the absorbance (OD) value (x) of the SVCV protein of the gradient dilution measured by the Bradford method is established, wherein the equation is that the value is 434.98x-163.01, and R is²Up to 0.995 (see fig. 9). Meanwhile, a TaqMan probe method qPCR-based method is adopted to determine corresponding gradient dilution SVCV copyThe point number is calculated, and then the correlation between the SVCV protein concentration and the SVCV copy number is established, and the linear regression equation of the two is that y is 3.48 multiplied by 10^-5x-3.26×10^-6(x is the concentration of SVCV protein in. mu.g/mL; y is the copy number of SVCV in copies/. mu.L), R²Up to 0.997 (see figure 10).

In order to verify the repeatability of the method, the established SVCV amplification culture and ultracentrifugation are adopted to combine with the density gradient centrifugation purification method to re-culture the amplification and purification SVCV, the concentration of the SVCV protein of the batch diluted by the gradient is determined by the Bradford method, the copy number of the SVCV diluted by the corresponding gradient is determined by the qPCR based on the TaqMan probe method, and the result shows that the protein concentration and the SVCV copy number of the SVCV purified again are equally and obviously positively correlated, and the linear regression equation of the SVCV and the SVCV is that the y is 2.52 multiplied by 10^-5x-3.64×10^-6(x is the concentration of SVCV protein in. mu.g/mL; y is the copy number of SVCV in copies/. mu.L), R²Up to 0.982 (see figure 11).

As the relevance between the copy number reflected by the QPCR method for detecting the SVCV nucleoprotein N based on the TaqMan probe method and the protein concentration of the SVCV directly determined by the Bradford method is established, the protein concentration and the copy number of the SVCV purified in different batches have obvious positive correlation.

Therefore, the invention establishes a simple and convenient SVCV quantitative method with high sensitivity and repeatability, and after establishing a regression equation between the protein concentration and the copy number of gradient dilution of SVCV of each batch of purified SVCV, the copy number of SVCV can be calculated by directly measuring the protein content of the purified SVCV and the regression equation in the subsequent research of viruses of the same batch.

SEQUENCE LISTING

<110> Shenzhen university

SHENZHEN ACADEMY OF INSPECTION AND QUARANTINE

<120> method for quantifying spring viremia of carp virus

<130> SZ1137-18P122149

<160> 7

<170> PatentIn version 3.3

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Claims (2)

1. A method for quantifying a spring viraemia of carp virus, said method not being used for the diagnostic treatment of a disease, characterized in that it comprises:

1) amplifying nucleoprotein N gene from spring viremia of carp virus to be detected, constructing recombinant plasmid, and establishing absolute quantitative SVCV virus copy number by taking the plasmid as a template based on a standard curve made by TaqMan probe method qPCR; the step 1) comprises the following steps:

1a) inoculating viruses to be detected to host cells, carrying out amplification culture, extracting and purifying a sample containing SVCV to prepare a sample solution to be detected;

1b) reverse transcription is carried out on the sample solution to be detected obtained in the step 1a) to extract cDNA of virus to be detected, and recombinant plasmid containing coding SVCV nucleoprotein N gene is prepared;

1c) taking the recombinant plasmid obtained in the step 1b) as a standard template, respectively carrying out real-time fluorescent quantitative PCR reaction by using a dye method and a Taqman probe method, and calculating the DNA copy number of the recombinant plasmid;

1d) taking the logarithm of the copy number of the plasmid as an abscissa and the Ct value as an ordinate to prepare a standard curve;

2) determining the protein concentration of the SVCV virus to be detected by using a Bradford method, and establishing a correlation with the copy number of the SVCV detected based on a qPCR method, wherein the step 2) comprises the following steps:

2a) preparing a protein standard curve by taking the bovine serum albumin diluted in a gradient manner as a standard substance;

2b) diluting the virus solution to be detected in a gradient manner, and calculating the protein concentration of the virus solution of each dilution gradient by using the OD value under the 595nm condition by using the Bradford method through the protein standard curve in the step 2 a);

2c) performing real-time fluorescence quantitative PCR reaction by taking diluted virus liquid as a template, performing reverse transcription on the virus into cDNA, and taking the reversed cDNA as the template;

2d) calculating the copy number of the virus liquid according to the standard curve made in the step 1 d);

2f) carrying out correlation analysis on the protein concentration measured in the step 2b) and the virus copy number measured in the step 2 d);

3) a quantitative method for determining the copy number of the SVCV virus by directly measuring the protein concentration when said SVCV virus is re-detected by the correlation establishment described in step 2), said step 3) comprising:

3a) amplifying and purifying the SVCV virus to be detected again;

3b) performing gradient dilution on the repurified virus solution, and determining the protein concentration of the virus solution of each dilution gradient by using a Bradford method;

3c) calculating the number of copies of the virus from the protein concentration determined in step 3b) on the basis of the correlation established in step 2 f).

2. A quantification method according to claim 1, comprising the following steps in sequence:

i) culturing EPC cells in an M199 culture medium, carrying out passage, inoculating viruses to be detected, continuously culturing until 90% of cells have cytopathic effect, and collecting cell and virus mixed liquor;

ii) repeatedly freezing and thawing the collected cell and virus mixed solution, and filtering to remove cell debris to obtain virus primary extract;

iii) centrifugally precipitating the virus primary extract, and re-suspending the virus sample by using a buffer solution to obtain a super-separation virus sample;

iv) preparing sucrose solutions with density gradients of 15%, 30%, 45% and 60%, adding the ultra-separated virus samples on the sucrose solutions with density gradients, centrifuging, and collecting virus distribution areas with liquid in 45% and 30% sucrose layers to prepare purified virus samples;

v) using SEQ ID NO.2 and SEQ ID NO.3 as primers, using a purified virus sample as an RNA template, and performing RT-PCR by using a reverse transcription kit to amplify cDNA of a nucleoprotein N gene;

vi) constructing a recombinant plasmid from the obtained cDNA and transforming into DH5 alpha;

vii) designing a qPCR primer and a Taqman probe of the recombinant plasmid, extracting the recombinant plasmid from the bacterial liquid to be used as a standard template, and performing real-time fluorescent quantitative PCR reaction by using a dye method and a Taqman probe method respectively;

the DNA copy number of the recombinant plasmid was calculated according to the following method:

molecular copy (. mu.L) ═ 6.02X 10²³) X (plasmid concentration X10)^-9) V (plasmid bp number × 660)

Taking the logarithm of the copy number of the plasmid as an abscissa and the Ct value as an ordinate to make a standard curve;

viii) diluting a series of concentration gradients by using BSA as a standard substance, then adding a Bradford reagent, measuring absorbance at 595nm by using an enzyme-labeling instrument after reaction, and taking the OD value of the absorbance as a horizontal coordinate and the protein concentration as a vertical coordinate to make a standard curve;

ix) diluting the purified SVCV virus solution in gradient, and then calculating the protein concentration of the virus solution of each dilution gradient by using the absorbance value under 595nm by using a Bradford method through the protein standard curve in the step viii);

x) taking diluted SVCV virus liquid as a template, carrying out reverse transcription on the virus to form cDNA, taking the reversed cDNA as the template, carrying out real-time fluorescence quantitative PCR reaction, and calculating the copy number of the virus liquid according to the standard curve made in the step vii);

performing correlation analysis on the protein concentration of SVCV measured by the Bradford method in the step ix) and the virus copy number measured by the RT-qPCR in the step x) to obtain a regression equation between the protein concentration and the copy number of the gradient dilution;

xi) for the same batch of virus, the copy number of SVCV was calculated by regression equations directly from subsequent studies by determining the protein content of the purified SVCV.

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