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TMV-CMV (Tobacco Mosaic Virus-Cucumber Mosaic Virus) double-virus colloidal gold quick detection test strip

A colloidal gold and virus technology, applied in the field of pathogen detection, can solve problems such as being expensive and unsuitable for tobacco seedling detection and use, and achieve the effects of simple operation, easy detection of a large number of samples, and timely and accurate diagnosis.

Active Publication Date: 2017-03-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Foreign commercial test strips are very expensive, the price is 50-60 yuan / sheet, which is not suitable for the detection of a large number of tobacco seedlings in production

Method used

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  • TMV-CMV (Tobacco Mosaic Virus-Cucumber Mosaic Virus) double-virus colloidal gold quick detection test strip
  • TMV-CMV (Tobacco Mosaic Virus-Cucumber Mosaic Virus) double-virus colloidal gold quick detection test strip
  • TMV-CMV (Tobacco Mosaic Virus-Cucumber Mosaic Virus) double-virus colloidal gold quick detection test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The isolation of embodiment 1 Guangdong tobacco area TMV, CMV virus strain

[0039] 1. From 2012 to 2014, from Nanxiong, Shixing, Ruyuan, Lechang in Shaoguan City, Guangdong Province, Lianzhou in Qingyuan City, Wuhua, Jiaoling, Dapu and Meixian in Meizhou City, etc. 29 Sampling was carried out on tobacco plants exhibiting common mosaic disease in tobacco fields in 3 towns and 33 villages. The sampling points covered all smoking areas in Guangdong Province. A total of 72 samples. Separate and identify them by biology and ELISA (enzyme-linked immunosorbent assay).

[0040] A total of 49 TMV isolates and 24 CMV isolates were isolated, all of which have been verified by ELISA. The results of biological identification showed that the collected TMV and CMV isolates belonged to their respective common strains.

[0041] 2. Amplify the full CP (coat protein) genes of the two viruses respectively. After the gel recovery of the PCR product, it was ligated with the pMD-18T vec...

Embodiment 2

[0045] Embodiment 2 prepares two kinds of CP gene prokaryotic expression specific proteins

[0046] CP-T-GD and CP-C-GD were respectively constructed on the pET30a-GST vector, transformed into BL21 strain, expressed in prokaryotic, and the expressed proteins were purified respectively.

[0047] 1. Preparation of CP-T-GD prokaryotic expression protein, relevant information is summarized in Table 1 below.

[0048] Table 1

[0049]

[0050] The specific method is as follows.

[0051] (1) Target gene sequence: according to the codon-optimized sequence of Escherichia coli, enzyme cutting sites EcoR I and Xho I are added at both ends. Synthesize the target gene into pUC57 vector.

[0052] (2) Connect the target gene to the pET30a-GST vector by enzyme digestion and ligation. The schematic diagram of the constructed recombinant vector is shown in the attached figure 1 shown.

[0053] Digestion of gene fragments: 43 μl recombinant plasmid, 1 μl EcoR I, 1 μl Xho I, 5 μl 10×Buffe...

Embodiment 3

[0094] Example 3 Construction of hybridoma cell lines expressing TMV and CMV two kinds of virus-specific monoclonal antibodies

[0095] Using the prokaryotic expression of specific proteins of the two CP genes obtained in Example 2, mice were immunized with the corresponding expressed proteins; a fusion test was carried out to obtain positive hybridoma cell lines producing specific monoclonal antibodies of the two viruses respectively.

[0096] 1. Construct a hybridoma cell line expressing TMV virus-specific monoclonal antibody, the method is shown in Table 3.

[0097] table 3

[0098]

[0099]

[0100] The specific method is as follows:

[0101] (1) immunity

[0102] 1) Initially immunize 4 SPF BALB / c female mice subcutaneously with "TMV" at the amount of 60ug protein / mouse, numbered as: 1, 2, 3, 4.

[0103] 2) Two weeks after the initial immunization, the subcutaneous booster immunization was given for the first time, with an immune dose of 30ug protein per mouse. ...

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Abstract

The invention discloses a TMV-CMV (Tobacco Mosaic Virus-Cucumber Mosaic Virus) double-virus colloidal gold quick detection test strip which consists of a sample pad, a colloidal gold pad, an NC membrane, water absorption filter paper and a back lining, wherein the colloidal gold pad is coated with a TMV specific antibody and a CMV specific antibody which are marked by colloidal gold; the two antibodies are respectively generated by hybridoma cell strains BALB / c-15-50 and BALBc-15-27; a detection line and a control line are arranged on the NC membrane; the detection line is coated with specific antigens of two viruses; the control line is coated with a colloidal gold marked second antibody. The test strip is quick, sensitive, accurate, low in cost and easy and convenient to operate particularly for detection of TMV and CMV in Guangdong tobacco-growing areas; simultaneous diagnosis of the two viruses is realized by one-time sampling; the TMV-CMV double-virus colloidal gold quick detection test strip is very suitable for on-site preliminary screening of a large batch of samples and has application value to actual production and a good popularization and application prospect.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection. More specifically, it relates to a TMV-CMV double virus colloidal gold rapid detection test strip. Background technique [0002] Tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are the main viruses infecting tobacco. The viral diseases they cause cause huge economic losses to the tobacco industry every year. Rapid and accurate qualitative detection of related viruses is one of the important means to prevent and control diseases and reduce losses in production. [0003] In scientific research, the detection methods of plant viruses mainly include biological detection methods (discrimination of symptom types, identification of hosts, etc.), electron microscope technology, serological detection methods (enzyme linked immunosorbent assay (ELISA), etc.) and molecular Biological detection methods (including PCR technology, nucleic acid probe technology, etc.), etc. Although th...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 阮小蕾
Owner SOUTH CHINA AGRI UNIV
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