Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof

Abstract

The invention disclose a dot- ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting the presence of the tomato yellow leaf curl virus in a plant as well as a reagent kit and an application thereof. A monoclonal antibody prepared for tomato yellow leaf curl virus coat protein is utilized to build the dotenzyme-Linked immunosorbent assay (dot-ELISA) method and the tissue printing ELISA method with optimal proportions, and the rapid-detection reagent kit is developed. The dot- ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting the presence of the tomato yellow leaf curl virus in a plant as well as the reagent kit and the application thereof are suitable for such solanaceae plants as tomato, capsicum, eggplant, tobacco, night shade and jimson weed; the situation of the presence of tomato yellow leaf curl virus in field vegetable samples is examined to detect the incidence of the virus disease and evaluate the occurrence and distribution and the prevalence trend of the tomato yellow leaf curl virus under the field condition; and the detection method has high sensitivity and good specificity, needs short time, is low in cost, and provides a technical support for rapid and large-scale detection of tomato yellow leaf curl virus.

Description

technical field [0001] The field of the invention belongs to the field of plant protection, in particular to the development and application of dot-ELISA and tissue imprint ELISA rapid detection method of tomato yellow leaf curl virus of tomato and its detection kit. Background technique [0002] Tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) causes tomato leaves to roll up or down, leaves yellow, leaf veins protruding, plant dwarfing, and in severe cases, tomato crop failure. According to reports, there are 15 kinds of pathogens causing tomato yellow leaf curl disease in China, among which tomato yellow leaf curl virus is the most widespread and most harmful in my country. Tomato yellow leaf curl virus belongs to the family Geminiviridae ( Geminiviridae ) Begomovirus ( Begomovirus ) virus, this genus virus can only be produced by Bemisia tabaci ( Bemisia tabaci ) are propagated in a persistent manner. [0003] Since tomato yellow leaf curl vi...

AI Technical Summary

Problems solved by technology

Although PCR detection technology is highly sensitive, it needs to go through the steps of genomic DNA extraction-PCR amplification-electrophoresis analysis, and even genome sequence determination. The cost of the test is high due to the lack of reagents, which is not conducive to the large-scale detection of viral diseases in the field

In addition, PCR technology is not easy to promote to non-professionals due to high false positives.

Although nucleic acid hybridization technology has strong specificity, the experimental operation is more professional and cumbersome. In addition to being highly dependent on instruments, it also has high requirements for reagents

Although traditionally used radioactive isotopes have high sensitivity, the reagents are harmful to the human body and the environment, and are not conducive to environmental protection.

Recently introduced alternative products such as digoxin reagents are free of radioactive contamination but are expensive. From the perspective of economy and practicality, it is difficult for nucleic acid hybridization technology to be applied to the detection of a large number of virus samples in the field.

Serological detection technology based on monoclonal antibody is a relatively simple detection method, which has the advantages of convenient operation and high sensitivity, but traditional serological methods such as triple antibody sandwich ELISA (TAS-ELISA), antigen-coated ELISA (ACP -ELISA) and other methods need to be carried out in a 48-well or 96-well enzyme-linked reaction plate. There are many experimental steps, and the reaction time of each step is long. Testing of Large Scale Samples

In the patent "Hybridoma cell line secreting monoclonal antibody against tomato yellow leaf curl virus and its application" (Chinese patent application number 201210004197.7) originally applied by our research group, the examples have briefly mentioned dot-ELISA and Tissue -blot ELISA detection method, but using this method in actual use, it is found that this method is only suitable for detecting viruses in one plant (ie tomato leaves), not suitable for other plants; in addition, due to the problem of plant sampling parts and reagent ratio As a result, the virus detection rate needs to be improved

In particular, the Tissue-blot ELISA kit mentioned in the examples consumes a lot of tomato leaves when applied in the field, the amount of reagents is large, and the cost is relatively high.

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