Tobacco mosaic virus Yunnan isolate TAS-ELISA test kit and its preparing method

A technology of tobacco mosaic virus and detection kit, which is applied in the preparation of test samples, measuring devices, and analysis materials, etc., and can solve the problems that have not yet been found, and the large-scale detection of plant viruses has not yet been discovered.

Inactive Publication Date: 2005-05-04
YUNNAN ACAD OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Judging from the existing literature reports at present, although the reports about the TAS-ELISA method for studying plant viruses have increased in recent years, the large-scale d...

Method used

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  • Tobacco mosaic virus Yunnan isolate TAS-ELISA test kit and its preparing method
  • Tobacco mosaic virus Yunnan isolate TAS-ELISA test kit and its preparing method

Examples

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Comparison scheme
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experiment example 1

[0145] The purpose of this experimental example is to study the routine detection method of the TAS-ELISA detection kit for TMV Yunnan isolates.

[0146]The researcher of the present invention studies the routine detection method of kit, sums up the detection method when TAS-ELISA detection kit of the present invention carries out tobacco virus detection, specifically as follows:

[0147] 1. TMV polyclonal antibody was diluted 1:500 times with coating buffer, added to the microtiter plate, 100ul per well, and placed at 37°C for 3h.

[0148] 2. Wash the plate 6 times with PBST, 3 times at a fast speed, 3 times at a slow speed, 3 minutes each time, and pat dry.

[0149] 3. Add 5-10 times the virus extraction buffer to the sample, grind the sample, add to the microplate plate, 100ul per well, add two wells for each sample, and place it overnight at 4°C. At the same time, positive, negative, and blank controls were set up in the same way.

[0150] 4. Wash the plate as above.

...

experiment example 2

[0159] The purpose of this experimental example is to study the rapid detection method of the TAS-ELISA detection kit for TMV Yunnan isolates.

[0160] The researcher of the present invention studies the quick detection method of kit, sums up the detection method when TAS-ELISA detection kit of the present invention carries out tobacco virus detection, specifically as follows:

[0161] Positive control, negative control, polyclonal antibody, monoclonal antibody, and enzyme-labeled antibody were prepared and stored in the same way as conventional detection methods.

[0162] The buffer was changed slightly:

[0163] ① Coating buffer: 4.52g in each kit, add 1000ml double distilled water for use

[0164] Na 2 CO 3 1.59g

[0165] NaHCO 3 2.93g

[0166] Dissolve in 1000ml double distilled water, adjust the pH value to 9.6.

[0167] ②PBST: 90.4g or 124.8g in each kit, add 8000ml double distilled water for use

[0168] Each 1000ml PBST contains:

...

experiment example 3

[0196] This experimental example verifies the discrimination of negative and positive, detection sensitivity and accuracy comparison.

[0197] When using a microplate reader to judge the detection results, record the OD value of each well of the microplate plate at a wavelength of 405nm by the microplate reader. Finally, when a large number of negative values ​​appear in the system detection, the determination of the whole system is invalid; the OD values ​​of the two wells measured by each sample should be basically the same, if the measured values ​​of the two wells differ greatly (generally refer to the OD value of two wells with the same dilution of the same sample) Exceeding the range of 0.5-1.5 times of its mean value), the enzyme plate assay is invalid. If the OD value of the positive control is lower than or close to the OD value of the negative control, the determination is also invalid. When the ratio of the OD value of the test sample to the negative control OD val...

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Abstract

This invention discloses a kin of tobacco mosaic virus yunnan outlier three antibody sandwich enzyme immune adsorbing rule detection kit. It includes case, enzyme standard board and liquid set in the case. The liquid comprises positive and negative contrast, enzyme standard antibody, buffer solution and substrate. It also includes multiple and single antibodies of tobacco mosaic virus yunnan outlier. The single and multiple antibodies take tobacco mosaic virus extracting solution with 20-30mg/ml virus concentration as immune antigen. They are prepared by immune injection method and single clone antibody method. The detection sensitivity of the detection kit in this invention can reach 0.01-0.001ng/ml, and it is 100-1000 times than indirect ELISA and DAS-ELISA detection sensitivity.

Description

technical field [0001] The invention relates to a biotechnology product, in particular to a detection kit for tobacco mosaic virus (Tobacco mosaicvirus, TMV) Yunnan isolate triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and a preparation method thereof. Background technique [0002] Plant viruses are important pathogens of crop virus diseases. With the development of plant virus-free seedling industrialization and the rise of modern facility agriculture, the detection and prevention and control technology of plant viruses has been paid more and more attention. At present, the detection technology of plant virus has four methods: serological technology, indicator plant determination, electron microscope detection, and molecular detection (including the detection of viral nucleic acid and protein). Various methods confirm each other, and at the same time, there are limitations in detection sensitivity, specialization, equipment and facility conditions, ...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N33/531G01N33/547
Inventor 张仲凯丁铭汪继玲李艳芳周雪平方琦
Owner YUNNAN ACAD OF AGRI SCI
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