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RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit for maize yellow mosaic viruses and application of kit

A technology of RT-LAMP and leaf virus, applied in the field of molecular biology and virology, to achieve the effect of reducing pollution rate, reliable results and high sensitivity

Active Publication Date: 2019-07-05
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of a low-cost detection reagent and detection method that can quickly detect maize yellow mosaic virus on the market for use by scientific research units and testing institutions, so that scientific research units can quickly extract the virus and quickly detect whether it is present in the sample. Infected by the virus in order to specify further precautions

Method used

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  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit for maize yellow mosaic viruses and application of kit
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit for maize yellow mosaic viruses and application of kit
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit for maize yellow mosaic viruses and application of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0048] (1) The design, screening and optimization process of the specific RT-LAMP primer set of maize yellow mosaic virus are as follows:

[0049] According to the maize yellow mosaic virus (MaYMV) cp gene reported on GenBank (the cut-off time for the selected sequence is August 1, 2018), DNAMAN8.0 was used for sequence comparison analysis, and the conserved region ( figure 1 ), use the online primer design software PrimerExplore rV4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design primers, and finally select the following primer sets (Table 1).

[0050] Table 1 MaYMV RT-LAMP detection primers

[0051]

[0052] (2) Taking the total reaction volume of 10 μL as an example to prepare the maize yellow mosaic virus RT-LAMP detection reaction system, the detection reaction system includes:

[0053] 1μL 10×ThermoPol Buffer; MgSO 4 (100mM) 0.52μL; dNTPs (10mM) 1.4μL; FIP primer (10mM) 1.2μL, BIP primer (10mM) 1.2μL; F3 primer (10mM) 0.2μL, B3 primer (10mM) 0.2μL; large ...

Embodiment 2

[0060] The present embodiment further determines the optimum dNT Ps concentration of MaYMV RT-LAMP reaction by experiment, as follows:

[0061] Other reaction conditions are the same as in Example 1. Different dNTPs concentrations (0.2mM; 0.6mM; 1.0mM; 1.4mM; 1.8mM) (final concentration of the reaction solution) are set in this example. figure 2 It can be seen that at the concentration of 1.4mM dNTPs, there are obvious trapezoidal waterfall bands, and the bands are not obvious at other concentrations. It shows that 1.4mM dNTPs is the best concentration in the RT-LAMP reaction system.

Embodiment 3

[0063] The present embodiment further determines the optimum Mg concentration of MaYMV RT-LAMP reaction by experiment, as follows:

[0064] When the concentration of dNTPs is 1.4mM, set different Mg 2+ Concentration (2mM; 4mM; 6mM; 8mM; 10mM) (reaction solution final concentration), other conditions are the same as in Example 1. Amplified by image 3 It can be seen that in Mg 2+ When the final concentration is 6-8mM, there are obvious trapezoidal waterfall-like bands, indicating that maize yellow mosaic virus can be specifically detected.

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Abstract

The invention discloses an RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit for maize yellow mosaic viruses. The kit comprises outer primers F3 and B3, inner primers FIP and BIP, RT-LAMP reaction solution and an RT-LAMP detection system formed by mixed solution of the RT-LAMP reaction solution and a specific RT-LAMP primer set. The kit can detect the maize yellow mosaic viruses simply and quickly, and overcomes the limitation of strong dependence of virus detection on instruments. The kit can be used for detecting the maize yellow mosaic viruses of maize andsorghum. In addition, the invention further discloses a detection method for detecting the maize yellow mosaic viruses by using the detection kit, the method can simply and quickly monitor whether amaize sample at a corn monitoring position is infected by the maize yellow mosaic viruses or not, and detection efficiency is improved.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and virology, in particular to an RT-LAMP detection kit for corn yellow mosaic virus and an application thereof. Background technique [0002] Maize yellow mosaic virus (MaYMV) is a new virus discovered and named in Yunnan and Guizhou, China in 2015. After the virus infects maize, it manifests as plant yellowing and mosaic symptoms. Whole-genome sequence analysis shows that the virus belongs to the family Luteoviridae (Family Luteoviridae) potato leafroll virus (GenusPolerovirus) virus, and has the highest homology with maize yellow dwarf virus-RMV (Maize yellow dwarf virus-RMV), and its homology sex as high as 73%. Under the electron microscope, the virion is equiaxed icosahedron without envelope. In 2017, the virus was also successively detected in maize in South America and maize, sugarcane (Saccharum spp) and tube grass (Rottboellia cochinchinensis) in Africa. It shows that the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/94
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/107
Inventor 王建光胡文丽陈穗云李小琴章彬
Owner YUNNAN UNIV
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