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Porcine senecavirus nucleic acid standard substance and application thereof

A virus nucleic acid and standard technology, applied in the field of swine Seneca virus nucleic acid standard, can solve the problems of lack of standard materials, uncalibrated, unclear source background, etc., to improve the quality and safety detection ability, stability and uniformity Good performance and guarantee the effect of smooth development

Inactive Publication Date: 2020-06-16
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on detection kits for swine Seneca is flourishing. Veterinary testing laboratories focus on the selection of serum detection kits, mainly imported, and domestic nucleic acid detection kits. However, domestic standards for these detection methods The materials are very scarce, and the testing institutions at all levels lack nucleic acid standard materials
Although the relevant scientific research units have more or less similar materials, their source background is often unclear, the preparation process is not perfect, and they have not been calibrated. Therefore, it is necessary to pass systematic research, inspection and calibration, and formulate corresponding technical regulations and Quality standards, in order to meet the basic requirements of standard substances

Method used

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  • Porcine senecavirus nucleic acid standard substance and application thereof
  • Porcine senecavirus nucleic acid standard substance and application thereof
  • Porcine senecavirus nucleic acid standard substance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The acquisition of the high porcine Seneca virus strain of embodiment 1 virus content

[0037] 1. Source of virus seed The virus seed used in the manufacture of this product is Seneca Valley virus SVV-HeNXX / swine / 2017 strain [because Seneca valley virus (Senecavalley virus, SVV) is also known as Seneca virus A (Senecavirus A, SVA ), so SVV and SVA all refer to the same virus in this application]; the virus species used for efficacy testing is the S-SVV strain, which is separated, identified, kept and supplied by the Henan Animal Disease Prevention and Control Center.

[0038] 2. Cell preparation Take out the frozen BHK21 cells from the working cell bank, thaw them in a 37°C water bath, centrifuge at 1000r / min for 15 minutes, discard the supernatant, resuspend with serum-free full suspension medium, and inoculate the bioreactor , set the temperature at 37° C., pH value 7.2, dissolved oxygen 50%, stirring speed at 100 r / min, perfusion speed at 60 r / min, and culture for 24...

Embodiment 2

[0043] The preparation of embodiment 2 porcine seneca virus nucleic acid standards

[0044] 1. Preparation of virus liquid

[0045] 1.1 Cell preparation Take out the frozen BHK21 cells from the working cell bank, thaw them in a water bath at 37°C, centrifuge at 1000r / min for 15 minutes, discard the supernatant, resuspend them with serum-free full suspension medium, and inoculate them into a bioreactor , set the temperature to 37° C., pH value to 7.2, dissolved oxygen to 50%, stirring speed to 100 r / min, perfusion speed to 60 r / min, and culture for 24 hours to 72 hours. The overgrown BHK21 cells were digested with trypsin and passaged at a concentration of 2×10 5 Viablecells / mL, placed in a 5% CO2 incubator for static culture.

[0046] 1.2 Virus cultivation and harvesting BHK21 cells that have grown into a good monolayer in the bioreactor, add the production seed virus SVA YRQ / 2016 with the preservation number CCTCC NO:V202013 at a ratio of 1% (V / V), and reset the temperature...

Embodiment 3

[0051] The inspection of embodiment 3 porcine seneca virus nucleic acid standards

[0052] The porcine seneca virus nucleic acid standard substance that embodiment 2 makes carries out following inspection:

[0053] 1. Physical property test

[0054] SVA standard substance should be colorless and transparent liquid.

[0055] 2. Virus inactivation test

[0056] Get 1mL of the standard solution prepared in Example 2 and inoculate it into the monolayer host cells. After culturing, no viral lesions appear. Quantitatively identified by PCR, the amount of virus after cultivation does not increase. Continuous blind passage for 3 generations shows no viral lesions. If there is no amplification curve identified by qPCR, the virus can be considered to be inactivated.

[0057] 3. Mycoplasma test

[0058] After dissolving the SVA standard substance, use the mycoplasma mycoplasma culture method to detect whether there is mycoplasma pollution. The test results are recorded in Table 1, a...

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Abstract

The invention relates to a porcine senecavirus nucleic acid standard substance and application thereof, and belongs to the technical field of virus standard substance preparation. A senecavirus strainwith the preservation number of CCTCC NO: V201767 is subjected to continuous passage and domestication to obtain an SVA strain with high virus content, the SVA strain is named as SVA YRQ / 2016, and the preservation number of the SVA strain is CCTCC NO: V202013. The virus content of each milliliter of the SVA strain is more than or equal to 10<12.5>TCID<50>. A preparation method comprises steps: preparing a porcine senecavirus solution from the SVA strain; inactivating, and adding a stabilizer and a protective agent; and carrying out uniformity evaluation, stability evaluation and valuation toobtain the porcine senecavirus nucleic acid standard substance which is accurate in valuation, has good traceability, stability and uniformity, can be used for SVA pathogenic microorganism nucleic acid detection and test mechanism evaluation and control test methods, and has huge quality control, quality arbitration and application values.

Description

technical field [0001] The invention relates to the technical field of virus standard product preparation, in particular to porcine seneca virus nucleic acid standard product and application thereof. Background technique [0002] Porcine Seneca virus disease is an infectious disease of pigs caused by Senecavirus A (SVA), which can cause piglets, fattening pigs, sows and boars nose, lip, hooves, abdomen Blisters, pustules, ulcers, etc. appear on the back and other parts, accompanied by lameness and difficulty in standing. The mortality rate of sick pigs can reach 30%-70%, and the production performance of sick pigs is greatly reduced. The effective detection of SVA is the basis of preventing and controlling porcine Seneca virus disease, and the accurate detection of SVA depends on high-quality SVA standard materials. At present, there is no institution or enterprise at home and abroad that produces SVA-related reference materials. In order to ensure the accuracy, comparabili...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/70C12Q1/6851C12R1/93
CPCC12N7/00C12N2770/32021C12Q1/6851C12Q1/701C12Q2531/113
Inventor 闫若潜班付国谢彩华王东方赵雪丽王淑娟王翠刘影方先珍刘梅芬马震原王华俊杨海波朱前磊张利平李蛟
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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