Method for detecting toxigenic pasteurellamultocida toxin formaldehyde inactivated efficacy by using Vero cells (African Green Monkey Kidney cells)

A technology for the inactivation of Pasteurella and formaldehyde, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. problem, to achieve the effect of ensuring safety

Active Publication Date: 2015-08-19
天康生物制药有限公司
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Problems solved by technology

Due to the different culture time of strains, individual differences in guinea pigs and personnel operating errors, false negatives may be caused. At the same time, the skin necrosis test of guine

Method used

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  • Method for detecting toxigenic pasteurellamultocida toxin formaldehyde inactivated efficacy by using Vero cells (African Green Monkey Kidney cells)
  • Method for detecting toxigenic pasteurellamultocida toxin formaldehyde inactivated efficacy by using Vero cells (African Green Monkey Kidney cells)
  • Method for detecting toxigenic pasteurellamultocida toxin formaldehyde inactivated efficacy by using Vero cells (African Green Monkey Kidney cells)

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[0013] Preparation of bacterial solution: inoculate the toxin-producing Pasteurella multocida into the brain-heart infusion medium, place it in a constant temperature shaking incubator at 36-38°C, and cultivate it at 200r / min for 12-18 hours, and harvest the bacterial solution.

[0014] Inactivation of the bacterial liquid: add formaldehyde to the toxigenic Pasteurella multocida bacterial liquid respectively according to the final concentration (v / v) of 0.2%, 0.25% and 0.3%, put in a constant temperature shaking incubator at 36~38°C, and set the temperature at 200r / min for 36, 48 and 60 hours respectively. Take 10ml of inactivated bacteria solution at each time point for use.

[0015] Guinea pig skin necrosis detection Take 0.1ml bacterial solution for each concentration and each time period, inject guinea pigs intradermally on the back, observe for 72 hours after injection, and measure the size of the skin necrosis area at the injection point. A necrotic area with a diamete...

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Abstract

The invention belongs to the field of biotechnology, particularly relates to a method for detecting toxigenic pasteurellamultocida toxin formaldehyde inactivated efficacy by using Vero cells (African Green Monkey Kidney cells), and comprises the following steps: step 1: preparing bacterium solution of toxigenic pasteurellamultocida toxin; step 2: preparing inactivated bacterium solution for ready use; step 3: preparing toxin solution of the toxigenic pasteurellamultocida toxin; step 4: after digesting and counting cultured monolayer Vero cells, diluting cell suspension by using MEM culture medium containing 2% of fetal calf serum, adding 100 mu l of MEM culture medium into each hole of a multi-hole experiment orifice; adding the toxin solution into the holes according to an equivalent times dilution method; further, adding 100 mu l of Vero cell suspension into each hole, uniformly mixing, placing in a CO2 incubator with the temperature of 36 to 38 DEG C for culturing and observing a result after seven days; determining cell drawing lesion as positive, bacteria agglomeration as suspicious, and no lesion as negative. By utilizing the method, whether the toxigenic pasteurellamultocida toxin is completely inactivated or inactivated to the greatest extent is more accurately detected, so that the safety of relevant vaccines are well ensured.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for detecting the formaldehyde inactivation effect of toxin-producing Pasteurella multocida toxin by using Vero cells. Background technique [0002] Toxigenic pasteurella multocida (T + Pm) is the main pathogen of porcine infectious atrophic rhinitis (Atrophicrhinitis, AR). Porcine atrophic rhinitis can cause turbinate atrophy in pigs, destroy the immune barrier of the nasal mucosa, and then cause other respiratory diseases, resulting in significant economic losses for the pig industry. Studies have shown that toxigenic Pasteurella multocida can cause progressive atrophic rhinitis, and the toxigenic Pasteurella multocida toxin is the main pathogenic factor. Pasteurella multocida is divided into 6 serotypes such as A, B, D, E, and F according to the capsule. It has been reported that T + Pm mainly produces D-type toxins, and also has A-type toxins, while T + Pm was mainly...

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 郝成武韩端何海贺笋李延涛何传雨何玉仙张淑琴
Owner 天康生物制药有限公司
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