A quantitative method for carp spring viremia virus

Abstract

The present invention amplifies the nucleoprotein gene N of SVCV through molecular biology techniques, and constructs recombinant plasmid pMD TM 18‑T‑SVCV‑N, using this plasmid as a template to establish an absolute quantitative SVCV copy number method based on the TaqMan probe method qPCR as a standard curve. At the same time, the protein concentration of SVCV was measured by Bradford method, and the correlation with the copy number of SVCV measured by the qPCR method was established. For the first time, a method for directly measuring protein concentration to determine the copy number of SVCV was established. This method can realize the simple and low-cost determination of the copy number of SVCV, and at the same time has important reference significance for the quantification of other viruses, and has important value in the related research of SVCV and other viruses.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a virus quantification method. Background technique [0002] Spring Viraemia of Carp (SVC for short) is a highly contagious epidemic disease of carps. The pathogen of the disease is Spring Viremia of Carp Virus (SVCV). SVCV causes the mortality rate of fish to be as high as more than 90%, which poses a great threat to the healthy breeding of freshwater fish. The Ministry of Agriculture promulgates "One, Two, and Three Types of Animal Epidemic Disease List (Aquatic Animal Epidemic Part)" (People's Republic of China Announcement of the Ministry of Agriculture, No. 1125) it is listed as a class of animal diseases. Moreover, the World Organization for Animal Health has also included it in the list of aquatic animal diseases (OIE, 2009, CHAPTER 2.3.8). In terms of its infectivity, it can be called "bird flu in aquatic animals" (this disease is listed as a class of animal diseases with b...

AI Technical Summary

Problems solved by technology

[0009] (1) A support film with good hydrophilicity must be used to make the sample structure and dyeing effect consistent, thereby ensuring the accuracy of quantitative analysis. It is difficult for ordinary copper mesh to meet the sample preparation requirements. The newly prepared carbon-Formvar (Formvar Or Collodion, Butvar and Pioloform and other substitutes) the copper mesh support film can ensure the uniform distribution of the adsorbed particles, but the effect will decrease significantly as the storage time prolongs;

[0010] (2) In the process of negative staining sample preparation, the degree of affinity between virus particles and the support membrane or dye will affect the quantity of adsorbed virus particles, the overall appearance of the virus structure and the change of osmotic pressure, which will affect the quantification;

[0011] (3) High equipment requirements;

[0012] (4) The sample preparation operation is relatively complicated, and requires a high level of operation for the experimenter

[0015] (1) The basis of the quantitative method of plaque formation test is that one infectious particle can form a plaque, but there is a possibility that multiple infectious particles can form a plaque at the same time, thus affecting the accuracy of quantification

A researcher mixed 10 kinds of gene-marked polioviruses (polioviruses) and conducted a plaque experiment. Among the 123 plaques formed, 6 plaques contained more than one virus. The study also found that low pH can lead to virus Aggregation of particles, which increases the probability of co-infection;

[0016] (2) The quantitative method of plaque formation test takes a long time. From the preparation of cells, virus inoculation, to the appearance of plaques, the whole process takes 3 days or more

[0024] In summary, the TaqMan probe method qPCR has the advantages of good specificity, high sensitivity, and good reproducibility. It is a relatively good method in the quantitative detection of viruses including SVCV, but due to its high cost , higher requirements for experimental technology and equipment

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