Method for detecting bovine viral diarrhea virus by virtue of indirect immunofluorescence

A technology of bovine viral diarrhea and immunofluorescence, which is applied in the field of detection of bovine viral diarrhea virus by indirect immunofluorescence method, can solve the problems of inability to judge the infectivity of BVDV particles, and achieve accurate detection effect

Inactive Publication Date: 2015-02-04
成都史纪生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology allows us to easily identify harmful microorganisms that may cause disease called boviditis or feline infectious peritonitis (FIP). It works by measuring how many different types of germs are found on dogs' blood cells during their incubations with these diseases. By doing this we aimed towards developing better ways to protect against such illnesses through immunization strategies.

Problems solved by technology

This patented technical problem addressed in this patents relates to the use of conventional techniques like virus separation or electrophoretic analysis that require long hours and skilled operators. These traditional approaches involve multiple steps involving different reagents and equipment, leading to increased risk from cross-contamination between samples.

Method used

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  • Method for detecting bovine viral diarrhea virus by virtue of indirect immunofluorescence

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Embodiment 1

[0025] Example 1: A kind of method utilizing indirect immunofluorescence method to detect bovine viral diarrhea virus, it comprises the following steps:

[0026] S1. Culture of MDBK cells

[0027] MDBK cells were cultured in cell culture flasks in MEM medium containing 10% horse serum at 37°C in 5% CO 2 Cultured in a constant temperature incubator, subcultured once every 2 days, and a single layer of MDBK cells was overgrown. After digested with trypsin, add growth medium to make cell suspension at 2×10 5 Inoculate in a 96-well plate at a density of 1 / ml, 100 μl per well, and place at 37°C in 5% CO 2 Cultured under the condition of 24h, the cells adhered to the wall and covered the bottom of the well after 24h; wherein, the growth solution was MEM medium containing 10% horse serum;

[0028] S2. Inoculation of BVDV

[0029] Inoculate BVDV with the MDBK cells cultured in step S1, add 2% horse serum to 200 μl per well, and culture for 72 hours, and set MDBK cells not in...

Embodiment 2

[0036] Example 2: A kind of method utilizing indirect immunofluorescence method to detect bovine viral diarrhea virus, it comprises the following steps:

[0037] S1. Culture of MDBK cells

[0038] MDBK cells were cultured in cell culture flasks in MEM medium containing 10% horse serum at 37°C in 5% CO 2 Cultured in a constant temperature incubator, subcultured once every 3 days, full of monolayer MDBK cells. After digestion with trypsin, add growth medium to make cell suspension at 4×10 5 Inoculate in a 96-well plate at a density of 1 / ml, 100 μl per well, and place at 37°C in 5% CO 2 Cultured under the condition of 24h, the cells adhered to the wall and covered the bottom of the well after 24h; wherein, the growth solution was MEM medium containing 10% horse serum;

[0039] S2. Inoculation of BVDV

[0040] Inoculate BVDV with the MDBK cells cultured in step S1, add 2% horse serum to 200 μl per well, and culture for 72 hours, and set MDBK cells not inoculated with BVD...

Embodiment 3

[0047] Example 3: A kind of method utilizing indirect immunofluorescence method to detect bovine viral diarrhea virus, it comprises the following steps:

[0048] S1. Culture of MDBK cells

[0049] MDBK cells were cultured in cell culture flasks in MEM medium containing 10% horse serum at 37°C in 5% CO 2 Cultured in a constant temperature incubator, subcultured once every 2.5 days, full of monolayer MDBK cells. After digested with trypsin, add growth medium to make cell suspension at 3×10 5 Inoculate in a 96-well plate at a density of 1 / ml, 100 μl per well, and place at 37°C in 5% CO 2 Cultured under the condition of 24h, the cells adhered to the wall and covered the bottom of the well after 24h; wherein, the growth solution was MEM medium containing 10% horse serum;

[0050] S2. Inoculation of BVDV

[0051] Inoculate BVDV with the MDBK cells cultured in step S1, add 2% horse serum to 200 μl per well, and culture for 72 hours, and set MDBK cells not inoculated with BV...

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Abstract

The invention discloses a method for detecting a bovine viral diarrhea virus by virtue of an indirect immunofluorescence, belonging to the technical field of biological quarantine. According to the method, the BVDV (bovine viral diarrhea virus) is bred by virtue of MDBK cells and is detected by virtue of the indirect immunofluorescence. The method specifically comprises the following steps: (S1) culturing the MDBK cells; (S2) inoculating the BVDV; and (S3) detecting the bovine viral diarrhea virus by virtue of the indirect immunofluorescence, namely carrying out washing, acetone fixation, primary antibody incubation, secondary antibody incubation and result determination. According to the method, the activities of BVDV particles can be rapidly and accurately detected, and polluted BVDV in swine fever live vaccines can be stably detected; the method is applied to the detection on the BVDV polluting bovine-origin raw materials in the vaccines, and a relatively efficient detection method for preparing pure swine fever live attenuated live vaccines is provided.

Description

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Claims

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Application Information

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Owner 成都史纪生物制药有限公司
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