Dot fluorescence immunoassay method for quantitatively detecting plant virus

Abstract

The invention discloses a dot fluorescence immunoassay method for quantitatively detecting a plant virus. The dot fluorescence immunoassay method comprises the following steps: grinding a sample to be tested, preparing a sampling solution of the detected sample, preparing a positive control sampling solution, a negative control sampling solution and a blank control sampling solution, measuring a virus particle average number d of the positive control sampling solution; respectively dotting 2.0-2.5 mu l of the sampling solution of the detected sample, the positive control sampling solution, the negative control sampling solution and the blank control sampling solution on a nitrocellulose membrane, closing for 0.8-1.2h after the nitrocellulose membrane is dried, sequentially adding a diluted primary antibody and a diluted secondary antibody, respectively reacting for 0.8-1.2h, washing the membrane for 3-4 times after each reaction is performed, wherein the washing of each time is performed for 5-6 min; and after slightly drying the membrane, detecting fluorescence intensity m in a fluorospectro photometer, and calculating a particle average number d of a virus to be tested in the sampling solution of the detected sample according to the following formula. A ratio of a value m of the positive control sampling solution to a value d of the positive control sampling solution is equal to a ratio of a value m of the sampling solution of the detected sample to a value d of the sampling solution of the detected sample. The dot fluorescence immunoassay method is capable of realizing sensitive, accurate, rapid and efficient quantitative analysis of the plant virus, and is high in popularization and application values.

Description

technical field [0001] The invention belongs to the technical field of quantitative detection of plant viruses, in particular to a fluorescent immunospot method for quantitative detection of plant viruses. Background technique [0002] Dot-immunobinding assay (DIBA) is a basic method for the detection of antigen-antibody immunological reaction and the identification of plant viruses using nitrocellulose film as a solid-phase carrier. This method has the advantages of high sensitivity, good accuracy, simple and quick operation, etc., but there are also some disadvantages: first, the judgment of negative and positive reactions can only be judged by visual inspection, so in samples with small color difference, it may cause misjudgment or Missed judgment; Secondly, although the color difference of the positive reaction reflects the amount of virus in the positive sample to a certain extent, it still cannot achieve true quantitative detection. Fluorescent labeling technology is ...

AI Technical Summary

Problems solved by technology

This method has the advantages of high sensitivity, good accuracy, simple and quick operation, etc., but there are also some disadvantages: first, the judgment of negative and positive reactions can only be judged by visual inspection, so in samples with small color difference, it may cause misjudgment or Second, although the color difference of the positive reaction reflects the amount of virus in the positive sample to a certain extent, it still cannot achieve true quantitative detection.