Indirect ELISA detection kit for B-type clostridium novyi

A technology of Clostridium novii and detection kit, which is applied in the field of indirect ELISA detection kits for Clostridium novyi of sheep B type, can solve the problem that the stability between batches cannot be guaranteed, the specificity is poor, the results vary greatly, etc. problem, to achieve the effect of effective detection, good stability and simple operation

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

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Problems solved by technology

When the prokaryotic expressed 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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  • Indirect ELISA detection kit for B-type clostridium novyi
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  • Indirect ELISA detection kit for B-type clostridium novyi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Establishment of indirect ELISA detection method:

[0047] Step 1. Preparation of coated antigen

[0048] Clostridium novyi was inoculated in the laboratory-made enrichment culture medium for Clostridium novyi, and the culture conditions were: cultivated in an anaerobic environment at 37°C for 3 days; the gas in the anaerobic environment The composition is: 80% nitrogen, 10% hydrogen, 10% carbon dioxide (both volume percentage); centrifuge at 10000-12000r / min for 3-5min, collect the bacteria, resuspend the bacteria with carbonate buffer, and make the bacteria suspension solution, which is the coating antigen.

[0049] Step 2. Coating of the microtiter plate

[0050] Take 100 μL / well of the bacterial suspension and add it to the microtiter plate, and place it at 4°C overnight. After coating, add PBST to the microtiter plate, 200 μL / well, shake gently for 3-5 minutes, discard the PBST in the microtiter plate, repeat washing 3 times, and pat dry the residual PBST in th...

Embodiment 5

[0088] Embodiment 5: the detection of clinical sample

[0089] Fifteen clinical blood samples and 5 blood samples from healthy sheep were collected from the diseased sheep farm. Separate the serum and measure its OD 450nm . The test results showed that the sera of the diseased sheep were all greater than the positive critical value, and the OD of the healthy sheep 450nm They are all less than the positive critical value, which is consistent with the actual disease status (the accuracy rate is 100%), which shows that the detection method established in this study is reliable. The specific test results are shown in Table 6.

[0090] Table 6: Detection results of clinical samples

[0091]

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Abstract

The invention discloses an indirect ELISA detection kit for B-type clostridium novyi. The indirect ELISA detection kit takes B-type clostridium novyi bacterium suspension as an envelope antigen, wherein the B-type clostridium novyi bacterium suspension is prepared by the following methods of inoculating B-type clostridium novyi into a culture medium to carry out bacterium-enrichment culture; centrifuging the cultured B-type clostridium novyi culture; and collecting bacterial cells, washing and resuspending the bacterial cells by using a carbonate buffer solution to prepare the B-type clostridium novyi bacterium suspension. The kit disclosed by the invention takes complete clostridium novyi bacterial cells as the envelope antigen, so that whether sheep serum contains an antibody to clostridium novyi or not can be detected at high sensitivity and high specificity and whether sheep are infected with a black disease or not can be judged accordingly. The indirect ELISA detection kit has the advantages that the detection result is high in specificity and good in repeatability, and a diagnosis can be quickly made and emergency treatment measures can be taken after the sheep catch the disease.

Description

technical field [0001] The invention relates to the technical field of Clostridium novyi detection, in particular to an indirect ELISA detection kit for Clostridium novyi type B in sheep. Background technique [0002] Sheep black plague, also known as "infectious necrotizing hepatitis", is an acute and highly fatal infectious disease caused by Clostridium novyi. Clostridium novyi can cause gas gangrene in humans, malignant edema in animals, macrocephaly in sheep and black plague in sheep, goats, cattle, horses, etc., with acute onset and high mortality, and is one of the zoonotic diseases A class of biological factors that are more harmful is an important disease that seriously endangers the breeding industry and brings huge economic losses to the development of animal husbandry in various countries. The analysis and detection of sheep black disease has not been paid attention to, but it is necessary to establish a rapid detection method for sheep black disease in the clini...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/558G01N33/569
CPCG01N33/535G01N33/558G01N33/56911G01N33/6854G01N2333/33G01N2469/20
Inventor 柴同杰孟德志韦良孟
Owner SHANDONG AGRICULTURAL UNIVERSITY
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