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Indirect ELISA detection kit for sheep clostridium putrificum

A detection kit and Clostridium putrefactive technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of animal disease that has not attracted considerable attention, and can not make a diagnosis quickly, to achieve effective detection, high sensitivity, and stability good sex effect

Active Publication Date: 2019-09-17
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the outbreak of sheep rapid disease in China can only be prevented and controlled initially, but it cannot be diagnosed quickly after the onset and emergency treatment measures are taken; while in foreign countries, the disease caused by Clostridium putrefaciens is mainly caused by human infection. Colon cancer, etc., has not attracted considerable attention in terms of animal pathogenesis

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  • Indirect ELISA detection kit for sheep clostridium putrificum
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Effect test

preparation example Construction

[0044](1) Preparation of coated antigen: the successfully identified Clostridium putrefaction colony was inoculated on the culture medium (the culture medium was composed of 36 parts of anaerobic meat jerky soup, 3 parts of glucose, 0.3 part of L-cysteine ​​hydrochloride , 7 parts of tryptone, and 1000 parts of water), under anaerobic conditions at 37°C (anaerobic environment gas composition volume ratio: nitrogen 78%, hydrogen 8%, carbon dioxide and helium in a volume ratio of 1:1 composition gas mixture 14%) for static culture for 12 to 24 hours, centrifuge the culture of Clostridium putrefaciens at 12000 rpm for 5 minutes, and collect the bacteria. Wash the cell pellet three times with PBS buffer, and resuspend the cell with carbonate buffer to make a bacterial suspension, which is the coated antigen.

[0045] (2) Antigen coating: dilute the Clostridium putrefaction suspension to OD with antigen coating solution (0.1M carbonate buffer, pH=9.6) 600nm When the value is 0.06,...

Embodiment 1

[0056] Embodiment 1: the establishment of indirect ELISA detection method

[0057] Determine the optimal coating concentration of the antigen, the optimal antigen coating condition, the optimal blocking condition, the optimal dilution factor of the serum to be tested, the incubation time of the serum to be tested, the optimal dilution concentration of the enzyme-labeled secondary antibody, and the enzyme-labeled secondary antibody. The best optimization scheme is determined as follows:

[0058] (1) Determination of the optimal coating dilution of the antigen

[0059] Dilute the Clostridium putrefaction suspension to OD with antigen coating solution (0.1M carbonate buffer, pH=9.6) 600nm The values ​​are 0.02, 0.04, 0.06, 0.08 and 0.1. The positive serum of Clostridium putrefaction is detected, and the absorbance value at 450nm is measured, and the optimal coating concentration of the antigen is determined according to the P / N value. Finally determine the optimal coating conce...

Embodiment 2

[0078] Embodiment 2: the establishment of indirect ELISA method standard curve

[0079] According to the operating procedures of the established indirect ELISA detection method for Clostridium putrefaction, the positive serum of Clostridium putrefaction was diluted to 7.32ng / mL by two times from 7500ng / mL, negative samples and blank wells were tested respectively, and the OD was measured after the reaction was completed. 450nm value. Take the positive serum concentration of Clostridium putrefaction as the abscissa, OD 450nm Draw a graph on the ordinate; according to the graph, select the best linear range, take the natural logarithm LN(x) of the concentration as the abscissa, OD 450nm A standard curve is plotted on the ordinate. The natural logarithm LN(x) of the standard serum concentration has a significant linear relationship with the detectable absorbance, and its correlation coefficient R 2 =0.9974, the linear equation is y=0.3622x-0.7971, the linear range is 14.65~187...

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Abstract

The invention discloses an indirect ELISA detection kit for sheep clostridium putrificum. The indirect ELISA detection kit is characterized in that a clostridium putrificum suspension is used as a coating antigen. The clostridium putrificum suspension is prepared by the following steps: inoculating a clostridium putrificum colony into a culture medium, and performing static culture for 12-24 hours in an anaerobic environment at the temperature of 37 DEG C; and centrifuging a clostridium putrificum culture obtained by culture, collecting the clostridium putrificum, performing washing, and re-suspending the clostridium putrificum by using a carbonate buffer solution to prepare the clostridium putrificum suspension. The kit disclosed by the invention can be used for detecting whether sheep serum contains an antibody of the clostridium putrificum in a high-sensitivity and high-specificity manner, and judging whether a sheep is infected with the clostridium putrificum or not according to the detection, so that after the sheep is sick, the diagnosis can be rapidly carried out and emergency treatment measures can be taken.

Description

technical field [0001] The invention relates to the technical field of Clostridium putrefaciens detection, in particular to an indirect ELISA detection kit for Clostridium putrefaciens. Background technique [0002] Clostridium putrefaciens, a Gram-positive anaerobic bacterium, is the main pathogen responsible for non-traumatic or spontaneous gas gangrene disease in humans, and the main cause of malignant edema disease in farm animals. Clostridium putrefaciens can easily cause rapid disease of sheep. This kind of disease has rapid onset and high mortality, which brings huge economic losses to animal husbandry. It has been reported that from 2002 to 2009, flocks of sheep in Northwest my country continued to suffer from the disease, and the fatality rate was as high as 60%. Since 2010, there have been successive cases of sheep disease in various parts of the country, such as Qinghai, Xinjiang, Guangxi, Guizhou, Hebei, Henan, and Gansu, with a fatality rate higher than 50%. M...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/558G01N33/569
CPCG01N33/535G01N33/558G01N33/56911
Inventor 柴同杰林静韦良孟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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