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Preparation method for ELISA detection kit of goat corynebacterium pseudotuberculosis antibodies

A detection kit and pseudo-tuberculosis technology, applied in the field of detection of Corynebacterium pseudotuberculosis, can solve the problems of large difference in results, poor terrer heterogeneity, and inability to guarantee the stability between batches and the like

Active Publication Date: 2015-12-23
NORTHWEST A & F UNIV
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AI Technical Summary

Problems solved by technology

When the prokaryotic expressed PLD protein is used as the coating antigen, the specificity is poor, and it will cross-react with other disease-positive sera
When using ultrasonically lysed bacteria as the coated antigen, the results of different batches of lysed bacteria are quite different, and the stability between batches cannot be guaranteed; and the bacteria are lysed to release internal proteins, which is prone to false positive test results

Method used

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  • Preparation method for ELISA detection kit of goat corynebacterium pseudotuberculosis antibodies
  • Preparation method for ELISA detection kit of goat corynebacterium pseudotuberculosis antibodies
  • Preparation method for ELISA detection kit of goat corynebacterium pseudotuberculosis antibodies

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preparation example Construction

[0011] The preparation method of the sheep pseudotuberculosis antibody detection kit that the present invention relates to comprises the following steps:

[0012] 1. Establishment of Indirect ELISA Detection Method

[0013] Step 1. Preparation of coated antigen

[0014] A single colony of Corynebacterium pseudotuberculosis was inoculated in 5mL of LB culture medium, and after expanded culture at 200-220rpm, 37°C for 48h, centrifuged at 10000-12000rpm for 3-5min, the bacteria were collected. The cells were washed three times with PBS, and the cells were resuspended with carbonate buffer to make a bacterial suspension. This is the coating antigen.

[0015] Step 2. Coating of the microtiter plate

[0016] Adjust the concentration of Corynebacterium pseudotuberculosis suspension on the spectrophotometer to make it OD 600 In the range of 0.060-0.090, take 100 μL / well of the bacterial suspension and add it to the microtiter plate, and place it at 4°C overnight. After coating, ...

specific Embodiment

[0049] 12 goats with clinical symptoms were collected from the sheep farm, 4 of which had typical symptoms of goat pus, 4 were diseased goats with normal viscera, and 4 were healthy goats. Separate the serum and measure its OD 450 . The test results show that the OD of sheep with body phenotype and visceral type 450 are greater than the positive critical value, the OD of healthy sheep 450 All were less than the positive critical value, which was consistent with the actual disease status, indicating that the detection method established in this study was reliable. The specific test results are shown in Table 6:

[0050]

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PUM

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Abstract

The invention relates to the biological technology field, and provides a preparation method for an ELISA detection kit of goat corynebacterium pseudotuberculosis antibodies. Complete corynebacterium pseudotuberculosis thalluses are employed as coating antigens, an indirect ELISA detection method is established, whether serum contains corynebacterium pseudotuberculosis antibodies is detected and whether a goat is infected with caseous lymphadenitis is determined. The positive and negative critical value is determined, and the detection kit has high specificity and repetition, can detect pseudotuberculosis antibodies in goat serum rapidly and effectively, and can be used for goat pseudotuberculosis diagnosis and epidemiological investigation.

Description

1. Technical field: [0001] The invention relates to the technical field of detecting Corynebacterium pseudotuberculosis, in particular to a preparation method of an ELISA detection kit for goat pseudotuberculosis antibody. 2. Background technology: [0002] Goat caseous lymphadenitis (Caseous Lymphadenitis, CLA) (also known as goat pseudotuberculosis) is caused by Corynebacterium pseudotuberculosis ( Corynebacterium pseudotuberculosis , C.p. ) caused by a chronic zoonotic disease characterized by enlarged lymph nodes, mainly infects goats and sheep, and is divided into two types: body phenotype and visceral type. Sick animals and latently infected animals can release a large amount of pathogens to the outside world through nasal secretions, excretions, and thick juice from ulcerated abscesses. Pathogens can infect healthy sheep through direct or indirect contact and cause soil, water, food, Pollution of pastures. Affected sheep have decreased milk production, reduced woo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56911G01N2333/34
Inventor 陈德坤霍宁宁刘鹤媛周铭
Owner NORTHWEST A & F UNIV
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