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IPMA antibody detection method of Lawsonia intracellularis

A technology for antibody detection and Lawsonia, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problems of not being able to reflect the true state of pig herd infection, and being unable to distinguish pig serum, etc., to achieve simple, intuitive and effective detection methods, and reduce detection Cost, specific effect

Pending Publication Date: 2021-09-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, compared with the wild strain of LI that causes pig herd infection, the strain in the commercial PPE vaccine has been artificially attenuated, and to a certain extent, it is different from the wild strain of naturally infected pigs. Vaccines are the source of detection, and often cannot reflect the truest state of infection in pig herds, let alone distinguish whether the antibodies in pig serum are from wild virus infection or the result of vaccine immunization

Method used

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  • IPMA antibody detection method of Lawsonia intracellularis
  • IPMA antibody detection method of Lawsonia intracellularis
  • IPMA antibody detection method of Lawsonia intracellularis

Examples

Experimental program
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Effect test

Embodiment 1

[0053] (1) McCoy cell subculture

[0054] Under sterile conditions, transfer the revived McCoy cells into a cell culture flask, add HDMEM culture solution containing 10% FBS, and place at 37°C, 5% CO 2 Cultivate in an incubator. After the cells are covered with a single layer, digest with 0.25% trypsin. Stop digestion when a small amount of cells fall off. Discard the digestion solution, add HDMEM cell culture medium containing 10% FBS, and repeat blowing several times until dispersed. For a single cell, pipette an appropriate amount of cell fluid into another cell bottle for subculture, and use it after the cells adapt to the culture environment.

[0055] (2) Culture of Lawsonia intracellulare

[0056] Will 1×10 5 When each / mL McCoy cells were cultured to a confluence of about 30%, the culture solution was discarded, and the Lawsonia intracellulare isolate prepared in Example 1 after 10-fold dilution of the LI inoculated culture solution was added, placed at 37°C, 8% o 2 ...

Embodiment 2

[0061] Determination of embodiment 2 IPMA optimal reaction conditions

[0062] 1. Determination of the optimal inoculation concentration of Lawsonia intracellulare and the working concentration of HRP-labeled goat anti-pig enzyme-labeled secondary antibody

[0063] The 96-well cell culture plate inoculated with McCoy cells was taken out from the incubator, the cell culture medium was discarded, and the Lawsonia intracellulare inoculated culture medium was used to inoculate the Lawsonia intracellulare stock solution (1×10 6 cells / mL) for doubling dilution, the dilution ratios were 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320, and 100 μL / well was added to 96 wells with cell monolayers. In the 96-well cell culture plate, add 100 μL of culture solution for Lawsonia intracellulare inoculation to each well of the first row of the 96-well cell culture plate, add 100 μL of undiluted LJS19051 stock solution to each well of the second row, and add 100 μL of undiluted LJS19051 stock solution ...

Embodiment 3

[0068] Specificity, sensitivity, repeatability test of embodiment 3 IPMA

[0069] The positive sera of porcine Escherichia coli, porcine salmonella, PEDV and TEGV identified and preserved by our laboratory were used for IPMA test to test the specificity of the method. The results showed that except the cytoplasm of McCoy cells inoculated with Lawsonia intracellulare was reddish-brown, the cytoplasm of cells inoculated with Escherichia coli, Salmonella, PEDV, and TGEV did not show color, indicating that the IPMA detection method established in this experiment has good specificity sex (see Figure 5 ). The standard positive sera of Lawsonia intracellulare were diluted 1:50, 1:100, 1:200, 1:400 and 1:800 times, and the IPMA test was performed to determine the sensitivity of the method. The results showed that when the Lawsonia intracellulare standard positive serum was diluted 1:50, 1:100; 1:200 and 1:400 times, the cytoplasm of McCoy cells infected by LI could still be detecte...

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Abstract

The invention discloses an IPMA antibody detection method of Lawsonia intracellularis. The method comprises the following steps: (1) culturing the Lawsonia intracellularis by using McCoy cells; (2) preparing an IPMA reaction plate: adding the Lawsonia intracellularis cultured in the step (1) into a cell plate containing McCoy cells for culturing, continuously culturing for 4-5 days, and then fixing, permeating and sealing in sequence; (3) adding to-be-detected serum into the IPMA reaction plate obtained in the step (2), respectively adding positive control serum and negative control serum at the same time, and incubating; adding a peroxidase-labeled secondary antibody, and incubating; and adding a developing solution, reacting and drying; and (4) observing under a microscope, and when the to-be-detected serum sample is positive of a lawsonia intracellularis antibody, staining the cytoplasm of the positive cell infected by the lawsonia intracellularis to be brownish red; and when a to-be-detected pig serum sample is negative in the lawsonia intracellularis antibody, enabling the cytoplasm of the McCoy cells infected by the lawsonia intracellularis to be not colored. The method has the advantages of high specificity, high sensitivity, simplicity in operation and the like.

Description

technical field [0001] The invention belongs to the technical field of Lawsonia intracellulare antibody detection, and in particular relates to an IPMA antibody detection method of Lawsonia intracellulare. Background technique [0002] Porcine proliferative enteropathy (PPE), commonly known as porcine ileitis, is an important pig disease caused by Lawsonia intracellularis (LI) infection, characterized by thickening of the ileum, cecum, and colon mucosa. Intestinal disease. Infected epithelial cells generally cannot develop into mature intestinal epithelial cells. As time goes by, mature intestinal epithelial cells are replaced by infected immature cells after aging, resulting in the infected animals not having normal absorption function, resulting in disease. The growth and development of pigs slowed down, the feed conversion rate decreased, and the cost of breeding increased, causing farmers to suffer serious economic losses. According to reports, in the United States, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56916
Inventor 范红结肖宁周红蔺辉星李剑男胡雨婷李敏雪
Owner NANJING AGRICULTURAL UNIVERSITY
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