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Method for detecting chrysanthemum B virus

A chrysanthemum and virus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of easy cross-contamination, high detection cost, complicated operation, etc., and achieve the effect of high specificity and easy identification.

Inactive Publication Date: 2009-06-10
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used polymerase chain reaction (PCR) detection method requires special equipment, high detection cost, and has the disadvantages of easy cross-contamination and cumbersome operation, and is not conducive to rapid on-site detection, so it is difficult to popularize and apply

Method used

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  • Method for detecting chrysanthemum B virus
  • Method for detecting chrysanthemum B virus
  • Method for detecting chrysanthemum B virus

Examples

Experimental program
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Embodiment 1

[0026] A method for detecting chrysanthemum B virus, which uses loop-mediated isothermal amplification reaction (LAMP) to detect chrysanthemum B virus.

[0027] The steps included in the method are as follows:

[0028] A. Prepare the reaction system in the Eppendoff tube of 1.5-2.0ml; The solvent used is redistilled water (ddH 2 O);

[0029] B. Incubate the reaction system to obtain an incubation product; the temperature of the incubation treatment is 65°C, and the time is 30-80min, preferably 60min; the incubation treatment is carried out in a metal constant temperature mixer ;

[0030] C. The incubation product is subjected to inactivation treatment to obtain a reaction product; the temperature of the inactivation treatment is 80° C., and the time is 5-15 minutes, preferably 10 minutes; the inactivation treatment is carried out in a metal constant temperature mixer It can also be carried out in a water bath or a metal bath;

[0031] D. detecting the reaction product and ...

Embodiment 2

[0038] This example is the composition of the reaction system described in Example 1. Include in this reaction system:

[0039] Betaine 0.6-1.4mol / L,

[0040] dNTP 0.25-2.5mmol / L,

[0041] Mg 2+ 2-12mmol / L,

[0042] Outer primers, inner primers; the concentration ratio of the inner primers and outer primers is 1:1-1:8;

[0043] Introduce Mg in this embodiment 2+ The method is: adding concentration is the MgCl of 2-12mmol / L in this reaction system;

[0044]The amount of the inner primer is 0.2 μmol / L, and the amount of the outer primer is 0.8 μmol / L; taking the system with a volume of 25 μl as an example, the system also includes: 1U Bst DNA polymerase large fragment 1 μl, 10×Bst Polymerse buffer 1μl, DNA template (21μg / ml concentration) 1μl, sufficient ddH 2 O;

[0045] The volume of the reaction system may also be 50 μl.

[0046] The DNA template is extracted from a chrysanthemum plant infected with B virus, and the preparation method of the DNA template is...

Embodiment 3

[0057] This example is a preferred scheme based on Example 2, and the quality and source of each component in the reaction system used are the same as those in Example 2. Include in this reaction system:

[0058] Betaine 0.6mol / L,

[0059] dNTP 0.4mmol / L,

[0060] Mg 2+ 2mmol / L,

[0061] Outer primers, inner primers; the concentration ratio of the inner primers and outer primers is 1:1;

[0062] Introduce Mg in this embodiment 2+ The method is: in this reaction system, adding concentration is the MgCl of 2mmol / L;

[0063] The amount of the inner primer is 0.2 μmol / L, and the amount of the inner primer is 0.2 μmol / L. Taking the system with a volume of 25 μl as an example, the system also includes: 1U Bst DNA polymerase large fragment 1 μl, 10×Bst Polymerse buffer 1μl, DNA template (21μg / ml concentration) 1μl, sufficient ddH 2 O;

[0064] The volume of the reaction system may also be 50 μl.

[0065] The primers and their sources used in this example are the sa...

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Abstract

The invention aims to provide a method for detecting chrysanthemum virus B (CVB) by utilization of loop-mediated isothermal amplification (LAMP). The method utilizes loop-mediated isothermal amplification (LAMP) to detect the chrysanthemum virus B (CVB). The method comprises the steps of preparation of a reaction system, incubation treatment, inactivation treatment and product detection in turn, wherein the reaction system comprises 0.6 to 1.4 mol / L of Betaine, 0.25 to 2.5 mmol / L of dNTP, 2 to 12 mmol / L of Mg<2+>, an outer primer and an inner primer, and the concentration ratio of the inner primer to the outer primer is 1:1-1:8. The method has the advantages that the method does not need a special instrument (such as a PCR instrument), and is quicker and more economic than the prior laboratory method; and the method is simple and convenient to identify, has high specificity, can judge whether a target fragment is amplified or not by observing with the naked eye or detecting the turbidity of deposit through a turbidity meter and an ultraviolet imaging system, so as to judge whether a sample plant to be detected is infected with the chrysanthemum virus B or not, and does not need to use a gel electrophoresis method for observation.

Description

Technical field: [0001] The invention relates to the detection field of plant viruses, in particular to a method for detecting chrysanthemum B virus, which uses a loop-mediated isothermal amplification reaction (LAMP) to detect chrysanthemum B virus. Background technique: [0002] Chrysanthemum is a traditional famous flower in China and one of the four major cut flowers in the world. However, due to the harm of viral diseases, the quality and output of chrysanthemum have declined, and the variety has degenerated very seriously. There are many types of viruses that harm chrysanthemums, and more than 20 have been reported. Chrysanthemum B virus (CVB) belongs to the genus Carnation latent virus and is one of the main viruses that harm chrysanthemum. The diseased chrysanthemums show mild mosaic disease or no symptoms on the leaves of chrysanthemums. On the susceptible varieties of chrysanthemums, obvious mosaic symptoms or necrotic spots can be formed, and brown scorched spots...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 黄丛林张秀海王永勤吴忠义梁宏霞李春华刘佳
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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