A tmv-cmv-pvy triple virus colloidal gold rapid detection test strip
A technology of TMV-CMV-PVY and detection test paper, which is applied in the field of pathogen detection, 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 timely and accurate diagnosis
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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, and 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 pro...
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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