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Porcine lawsonia intracellularis IPMA antigen detection method and application thereof

A technology for antigen detection and Lawsonia, applied in chemical instruments and methods, immunoassays, biological tests, etc., can solve problems such as false negatives, low content of Lawsonia intracellulare, and limited

Pending Publication Date: 2020-10-27
NANJING AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although PCR can realize the rapid detection of clinical samples, it is limited to the detection of fecal samples during the infection period
In addition, due to the low content of Lawsonia intracellulare in feces and the presence of PCR inhibitors, it may cause some false negatives
The interpretation of IFA results requires an expensive fluorescent microscope, and the test results may also be false negative. Failure to detect does not mean that there is no LI, and it is difficult to promote it in all laboratories.

Method used

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  • Porcine lawsonia intracellularis IPMA antigen detection method and application thereof
  • Porcine lawsonia intracellularis IPMA antigen detection method and application thereof
  • Porcine lawsonia intracellularis IPMA antigen detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: Prokaryotic expression and purification of SodC protein

[0071] (1-1) Using the attenuated vaccine culture of Lawsonia intracellularis suis as a template, design a pair of specific primers SF / SR for amplification:

[0072] SF: 5'-GGATCCATGGAATAAAACAGAGTATAGG-3' (SEQ ID NO.1);

[0073] SR: 3'-CTCGAGCTAGTTTGGTATAACACCAC-5' (SEQ ID NO. 2);

[0074] (1-2) Use pGex-6p-1 as the carrier, digest pGex-6p-1 with SacI and XhoI, connect the amplified product of step (1), construct the pGex-6p-1-sodc recombinant plasmid, transform BL21, and obtain the recombinant Bacteria pGex-6p-1-sodc / BL21;

[0075] Inoculate the recombinant strain pGex-6p-1-sodc / BL21 into 3ml of LB liquid medium containing ampicillin and chloramphenicol at a ratio of 1:100, culture at 37°C with shaking at 180rpm until OD 600nm When the value is about 0.5, add a final concentration of 0.5mMIPTG, induce at 16°C, 90rpm for 18h, centrifuge at 8000rpm, 4°C for 10min to collect the cells, wash the cel...

Embodiment 2

[0077] Embodiment 2: the preparation of the polyclonal antibody of SodC protein and Western-blot analysis

[0078] The purified SodC protein was mixed with an oil adjuvant (ISA206 oil adjuvant) at a ratio of 1:1, emulsified and prepared as an immunogen, and the New Zealand white rabbits were immunized by multi-point subcutaneous injection on the back, with an immunization dose of 1 mg / rat. On the 14th and 21st days after immunization, booster immunization was given once, and the immunization dose and immunization route were the same as the first immunization. On the 7th day after the third immunization, blood was collected from the ear vein of the rabbit, and the serum titer was determined by conventional ELISA method. The coating concentration of SodC protein was 20ug / ml, and the serum to be tested was diluted from 1:100 to 1:100. ×2 30 , the dilution factor of HRP-labeled goat anti-rabbit IgG is 1:5000, and the OD is determined after TMB color development 450nm The absorba...

Embodiment 3

[0079] Example 3: Cultivation of Lawsonia intracellulare

[0080] (1) Take out the McCoy cells from liquid nitrogen, put them into 37°C water to melt quickly, add the melted cells into DMEM high glucose medium containing 10% fetal bovine serum, 37°C, 5% CO 2 Cultivate in an incubator for 24 hours. After the cells are covered with a single layer, discard the culture medium, wash with PBS for 3 times, digest with 0.25% trypsin in mass ratio, stop digestion when a small amount of cells fall off, and add FBS containing 10% in volume ratio The DMEM cell culture medium was repeatedly blown several times to disperse into single cells, and an appropriate amount of cell liquid was sucked into another cell bottle for subculture.

[0081] (2) When McCoy is cultured to a density of about 30%, discard the DMEM cell culture solution containing 10% FBS in volume ratio, and add Lawsonia intracellulare after 10-fold dilution of the inoculated culture solution of Lawsonia intracellulare. For a...

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Abstract

The invention relates to a porcine lawsonia intracellularis immunoperoxidase monolayer assay (IPMA) antigen detection method. The rabbit anti-lawsonia intracellularis SodC protein polyclonal antibodyis prepared by taking purified recombinant SodC protein as immunogen, and the titer and the reactogenicity are detected. The lawsonia intracellularis IPMA antigen detection method is established by taking lawsonia intracellularis positive bacteria as a coating antigen and taking a prepared polyclonal antibody as a primary antibody. The method has good specificity, sensitivity and repeatability, expensive instruments such as a fluorescence inversion microscope, a microplate reader and a PCR instrument are not needed, operation is easy and convenient, judgment is more visual, the dyed microporous plate can be stored for a long time, the method is suitable for screening of a large number of samples, and a new means is provided for positioning and detection of lawsonia intracellularis in infected cells.

Description

technical field [0001] The invention belongs to the technical field of bacteria detection, and in particular relates to a Lawsonia intracellulare IPMA antigen detection method and application thereof. Background technique [0002] Porcine proliferative enteritis (PPE), also known as porcine ileitis, is an infectious intestinal disease caused by Lawsonia intracellularis (LI). The bacterium usually infects nursery pigs, and the lesions mainly occur in the terminal ileum. Clinically, it is characterized by hyperplasia and thickening of the mucous membrane, which can cause growth retardation and a decrease in the proportion of feed to meat in pigs, causing serious economic losses to pig farms. The disease presents three forms: acute type, subacute type and chronic type. The subacute type is the main clinical type, which generally does not cause the death of infected pigs. The disease was first reported in 1931, widely seen in some countries in Europe, North America, Australia a...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/573G01N33/569G01N33/531G01N21/84G01N21/78C07K16/12C07K16/06
CPCG01N33/581G01N33/573G01N33/56911G01N33/531G01N21/78G01N21/84C07K16/1203C07K16/06G01N2333/90283G01N2469/10
Inventor 范红结李敏雪肖宁李剑男
Owner NANJING AGRICULTURAL UNIVERSITY
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