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TMV single colloidal gold quick detection test paper strip

The technology of colloidal gold and colloidal gold pad is applied in the field of pathogen detection, which can solve the problems of being expensive and unsuitable for the detection of tobacco seedlings, and achieve the effects of simple operation, easy detection of a large number of samples, and harmless to human body.

Pending Publication Date: 2017-03-15
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 single colloidal gold quick detection test paper strip
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  • TMV single colloidal gold quick detection test paper strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation of TMV virus strain in Guangdong tobacco area

[0034] 1. From 2012 to 2014, 29 counties from 9 counties including Nanxiong, Shixing, Ruyuan, Lechang in Shaoguan City, Guangdong Province, Lianzhou in Qingyuan City, Wuhua, Jiaoling, Dapu and Meixian in Meizhou City Samples were taken from tobacco plants exhibiting common mosaic disease in tobacco fields in 3 towns and 33 villages. The sampling points cover all the smoking areas in Guangdong Province. A total of 72 samples. They were separated and identified by biology and ELISA (enzyme-linked immunosorbent assay) respectively.

[0035] A total of 49 TMV isolates were isolated, all of which have been verified by ELISA. The results of biological identification showed that the collected TMV isolates belonged to ordinary strains.

[0036] 2. Amplify the full CP (coat protein) gene of TMV virus. The PCR product was recovered from the gel and ligated with the pMD-18T vector to transform E. coli DH5α. The pos...

Embodiment 2

[0040] Example 2 Preparation of Prokaryotic Expression Specific Protein of CP Gene

[0041] 1. Prepare CP-T-GD prokaryotic expression protein, construct CP-T-GD on pET30a-GST vector, transform BL21 strain, perform prokaryotic expression, and purify the expressed protein.

[0042] The relevant information is summarized in Table 1 below.

[0043] Table 1

[0044]

[0045] 2. The specific method is as follows.

[0046] (1) Target gene sequence: The sequence is optimized according to the E. coli codon, with restriction sites EcoR I and Xho I added at both ends. Synthesize the target gene into pUC57 vector.

[0047] (2) Connect the target gene to the pET30a-GST vector through restriction digestion and ligation. The schematic diagram of the constructed recombinant vector is shown in the attached file. figure 1 Shown.

[0048] Gene fragment digestion: 43μl recombinant plasmid, 1μl EcoR I, 1μl Xho I, 5μl 10×Buffer, 37℃ overnight reaction. (Sepharose DNA Recovery Kit, BPI).

[0049] Vector digest...

Embodiment 3

[0081] Example 3 Construction of a hybridoma cell line expressing TMV virus specific monoclonal antibody

[0082] The CP gene obtained in Example 2 was used to express a specific protein in prokaryotic cells, and mice were immunized with the expressed protein; a fusion test was performed to obtain a positive hybridoma cell line producing a specific monoclonal antibody to the TMV virus.

[0083] 1. The specific method is shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] 2. The specific method is as follows:

[0088] (1) Immunity

[0089] 1) Use "TMV" to immunize 4 SPF BALB / c female mice subcutaneously at the amount of 60ug protein / mouse, numbered: 1, 2, 3, 4.

[0090] 2) Two weeks after the initial immunization, the first booster immunization subcutaneously with 30ug protein / mouse.

[0091] 3) Two weeks after the first booster immunization, the second booster immunization subcutaneously, the immunization amount is 30ug protein / mouse.

[0092] 4) Two weeks after the second booster...

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Abstract

The invention discloses a tobacco mosaic virus (TMV) single colloidal gold quick detection test paper strip, which is composed of a sample pad, a colloidal gold pad, an NC membrane, a water absorption filter paper and a back lining. The colloidal gold pad is coated by a TMV specific antibody marked by colloidal gold. The antibody is secreted by hybridoma cell strain BALB / c-15-50; a detection line and a control line are arranged on the NC membrane, wherein the detection line is coated with specific antigens of three viruses, and the control line is coated with a secondary antibody marked by the colloidal gold. The test paper strip is mainly used for detection on the TMV in tobacco planting regions in Guangdong, is quick, sensitive, accurate and low-cost, has simple operations and achieves simultaneous diagnosis of multiple viruses through one-time sample collection, and is very suitable for in-site primary screening of a large amount of samples. The test paper strip has great application value in practical production and has good promotion and application prospects.

Description

Technical field [0001] The invention belongs to the technical field of pathogen detection. More specifically, it relates to a test strip for rapid detection of TMV single virus colloidal gold. Background technique [0002] Tobacco mosaic virus (TMV) is one of the main viruses that infect tobacco. The viral disease it causes causes huge economic losses to the tobacco industry every year. Quick and accurate qualitative detection of related viruses is one of the important methods 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 (symptom type identification, host identification, etc.), electron microscopy, serological detection methods (Enzyme linked immunosorbent assay (ELISA), etc.) and molecular Biological detection methods (including PCR technology, nucleic acid probe technology, etc.), etc. Although electron microscopy is an effective method to di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569G01N33/558
CPCC07K16/10G01N33/558G01N33/56983
Inventor 阮小蕾
Owner SOUTH CHINA AGRI UNIV
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