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TMV-CMV-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip

A TMV-CMV-PVY and colloidal gold technology, applied in the field of pathogen detection, can solve the problems of being expensive and not suitable for the detection of tobacco seedlings, and achieve the effects of simple operation, easy detection of a large number of samples, and convenient portability

Active Publication Date: 2017-05-10
SOUTH CHINA AGRI UNIV +2
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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-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip
  • TMV-CMV-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip
  • TMV-CMV-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The isolation of embodiment 1 TMV, CMV, PVY virus strain in Guangdong tobacco-growing area

[0045] 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).

[0046] A total of 49 TMV isolates, 24 CMV isolates, and 13 PVY isolates were isolated, all of which have been verified by ELISA. The results of biological identification showed that the collected isolates of TMV, CMV and PVY all belonged to their common strains.

[0047] 2. Amplify the full CP (coat protein) genes of the three viruses respectively. After the gel recovery of the PCR product...

Embodiment 2

[0051] Embodiment 2 prepares three kinds of CP gene prokaryotic expression specific proteins

[0052] CP-T-GD, CP-C-GD, and CP-P-GD were respectively constructed on the pET30a-GST vector, transformed into BL21 strain, and prokaryotically expressed, and the expressed proteins were purified respectively.

[0053] 1. Preparation of CP-T-GD prokaryotic expression protein, relevant information is summarized in Table 1 below.

[0054] Table 1

[0055]

[0056]

[0057] The specific method is as follows.

[0058] (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.

[0059] (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.

[0060] Digestion of gene fragments: 43 μl recombinant plasmid, 1 μl EcoR...

Embodiment 3

[0110] Example 3 Construction of hybridoma cell lines expressing TMV, CMV, and PVY three kinds of virus-specific monoclonal antibodies

[0111] Using the prokaryotic expression of specific proteins of the three 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 three viruses respectively.

[0112] 1. Construct a hybridoma cell line expressing TMV virus-specific monoclonal antibody, and the method is shown in Table 4.

[0113] Table 4

[0114]

[0115]

[0116] The specific method is as follows:

[0117] (1) immunity

[0118] 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.

[0119] 2) Two weeks after the initial immunization, the subcutaneous booster immunization was given for the first time, with an immune dose of 30ug prot...

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Abstract

The invention discloses a TMV-CMV-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip, which is composed of a sample pad, a colloidal gold pad, an NC membrane, a piece of water-absorbent filter paper and a backing, wherein a colloidal gold labeled TMV specific antibody, a CMV specific antibody and a PVY specific antibody are enveloped on the colloidal gold pad, and the three antibodies are secreted from hybridoma cell strains BALB / c 15 50, BALBc 15 27 and BALBc 15 8; a detection line and a control line are arranged on the NC membrane; specific antigens of three viruses are enveloped on the detection line; and a colloidal gold labeled second antibody is enveloped on the control line. The test strip is especially suitable for detecting the TMV, CMV and PVY in Guangdong tobacco-growing areas, and the test strip is rapid, sensitive, accurate, low in cost and convenient to operate; the test strip is capable of simultaneously diagnosing various viruses by conducting sampling once; the test strip is quite suitable for in-situ preliminary screening of a great batch of samples; and the test strip, in actual production, has a high application value, and the test strip has 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-PVY triple virus colloidal gold rapid detection test strip. Background technique [0002] Tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY) 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 technolog...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/532G01N33/543G01N33/558G01N33/569G01N33/577
CPCC07K16/10G01N33/532G01N33/543G01N33/558G01N33/56983G01N33/577
Inventor 阮小蕾邓海滨王晓宾
Owner SOUTH CHINA AGRI UNIV
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