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Method for detecting animal pathogenic microorganisms and special protein chip thereof

A pathogenic microorganism and protein chip technology, which is applied in the detection method of animal pathogenic microorganisms and its special protein chip field, can solve the problems of poor detection effect, difficult protein purification, easy inactivation, etc., achieve good application prospects, improve detection efficiency, The effect of saving testing cost

Inactive Publication Date: 2010-01-20
INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty of protein purification and its easy inactivation during operation, the development of protein chip technology is obviously lagging behind that of gene chips. Currently, protein chips used for microbial detection have cross-reaction phenomena, and the detection effect is not good.

Method used

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  • Method for detecting animal pathogenic microorganisms and special protein chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Preparation of Microbial Protein Antigen Chips

[0020] Select four of the bacteria that must be excluded in the national standard (GB14922.2-2001) for specific pathogen-free (SPF) grade mice: Salmonella Typhimurium, Pasteurella pneumotropica, Pseudomonas tuberculosis Yersinia pseudotuberculosis, Streptobacillus moniliformis. Through multiple western blot experiments, 5 specific antigens were selected for each bacterium, and these antigens were prepared by bacterial lysis, ion exchange chromatography, SDS-PAGE gel recovery electrophoresis and other methods, a total of 20 kinds. After the antigen was properly diluted with phosphate buffered saline (PBS, pH 7.4), the antigen was spotted on the aminated glass slide with the AD3200 chip spotting instrument of BioDot Company, and each antigen was spotted three times, and then the spot The good protein chip was placed in a wet box overnight at 4°C; the next day, the chip was placed in a wet box to equilibrate at room tempera...

Embodiment 2

[0022] Serological detection of mice infected with Salmonella Typhimurium

[0023] The mouse serum infected with Salmonella typhimurium was diluted with 0.5% bovine serum albumin component V-PBS (BSA-PBS) at a volume ratio of 1:100, hybridized with the microbial protein antigen chip prepared in Example 1, and incubated at 37°C for 1h , and then wash the chip with PBS 3 times, 3min / time, to wash away unbound antibodies. Then hybridize the chip with the labeled secondary antibody, the labeled secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG diluted with PBS at a volume ratio of 1:200, incubated at 37°C for 1 h, and then washed with PBS Chip 3 times, 3min / time. Finally, the chip was developed with 3,3-diaminobenzidine (DAB), dehydrated, and sealed. A microarray scanner was used to scan the color development results of the chip, and software was used to analyze the scanning results. The results showed that Salmonella typhi antigens all produced strong po...

Embodiment 3

[0025] Serological detection of mice infected with Pasteurella pneumotropica

[0026] The mouse serum infected with Pasteurella pneumophila was diluted with 0.5% BSA-PBS at a volume ratio of 1:100, and then hybridized with the microbial protein antigen chip prepared in Example 1, and the hybridization experiment was carried out according to the operation method of Example 2. The results showed that the antigens of Pasteurella pneumophila produced strong positive signals, while the antigens of other bacteria had no signals.

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Abstract

The invention belongs to the field of biomedicine, providing a method for detecting animal pathogenic microorganisms and a special protein chip thereof. The detecting method essentially comprises the following steps of: a. preparing the protein chip, selecting a plurality of different groups of specific antigen of many kinds of pathogenic microorganisms by means of an experiment to be fixed on a properly modified carrier; and b. detecting: reacting blood serum of a animal to be detected with the protein chip, hybridizing with a marked secondary antibody, detecting a hybrid positive signal in a proper detection way, and obtaining a hybrid result of the blood serum and the chip by means of scanning and analyzing, wherein the blood serum infected with the different pathogenic microorganisms can show different positive signal combination modes after the reaction, so as to ensure whether the animal to be detected is infecting or is infected with the pathogenic microorganisms marked on the protein chip. The invention can simultaneously detect many kinds of the pathogenic microorganisms, improves detecting efficiency, selects many groups of different antigen with a certain specificity aiming at each pathogenic microorganism, leads the detection to have good specificity, and has good application prospect in detecting the animal pathogenic microorganisms.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for detecting animal pathogenic microorganisms and a special protein chip thereof. Background technique [0002] Animals are closely related to human life, production and scientific research work. It is of great significance to detect the microorganisms carried by animals and ensure the quality of animals to protect human health, promote economic development and ensure the accuracy of animal experiments. [0003] The traditional and currently most widely used microbial detection method is to conduct colony, individual morphology, and physiological and biochemical identification of microorganisms through isolation and culture. This method has a large workload, takes a long time, and has relatively high requirements for the experimental operator. [0004] With the development of modern science, some new technologies are applied to microbial detection. The polymerase chain reac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/531
Inventor 秦川张连峰林树柱李万波
Owner INST OF LAB ANIMAL SCI CHINESE ACAD OF MEDICAL SCI
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