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MP (movement protein)-gene-based real-time fluorescence polymerase chain reaction (PCR) detection method for cucumber green mottle mosaic virus (CGMMV), and probe and primer combination

A green mottled flower, real-time fluorescence technology is applied in the field of probe and primer combinations for detection, which can solve the problems of no good improvement in detection sensitivity, restrictions on popularization and application, and increase in cost, and achieves high sensitivity, improved sensitivity, and improved efficiency. Effect

Inactive Publication Date: 2013-04-24
张卫东 +6
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Problems solved by technology

Although time and steps are saved in the RNA extraction process, this method relies on highly sensitive antibodies, which increases the cost and does not improve the detection sensitivity, so its popularization and application are limited

Method used

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  • MP (movement protein)-gene-based real-time fluorescence polymerase chain reaction (PCR) detection method for cucumber green mottle mosaic virus (CGMMV), and probe and primer combination
  • MP (movement protein)-gene-based real-time fluorescence polymerase chain reaction (PCR) detection method for cucumber green mottle mosaic virus (CGMMV), and probe and primer combination
  • MP (movement protein)-gene-based real-time fluorescence polymerase chain reaction (PCR) detection method for cucumber green mottle mosaic virus (CGMMV), and probe and primer combination

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Embodiment Construction

[0033] The present invention will be described in detail below. The real-time fluorescent PCR detection method for cucumber green mottle mosaic virus based on the MP gene comprises the following steps:

[0034] 1. Select the test material. The virus samples used for testing in the present invention are shown in Table 1.

[0035] Table 1 Virus samples for testing

[0036]

[0037] The main reagents and instruments used in the present invention are as follows: PCR amplification reagents are provided by Shanghai Sangong Bioengineering Technology Co., Ltd.; primers and probes are synthesized by Bao Biological Engineering (Dalian) Co., Ltd.; other reagents are operated by ordinary molecular biology experiments Regulation. Fluorescent PCR instrument: Roche LightCycler 480 fluorescent quantitative PCR instrument; desktop high-speed centrifuge Eppendorf 5415D.

[0038] 2. Take the sample to be tested and extract the total RNA from the plant tissue.

[0039...

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Abstract

The invention discloses a MP (movement protein)-gene-based PCR (polymerase chain reaction) detection method for performing rapid molecular identification on cucumber green mottle mosaic virus (CGMMV), and a probe and primer combination applied to the method. The sequences of primers and a probe include forward primer MMV-F1, downstream primer MMV-R1 and fluorescence probe MMV-1. The method comprises the following steps of: (1) extracting total RNA (Ribose Nucleic Acid) of plant tissue from a sample to be tested; (2) introducing the downstream primer MMV-R1 for implementing synthesis of a reverse transcription template cDNA with taking the total RNA as a template; (3) performing real-time fluorescence PCR amplified reaction when the forward primer, the downstream primer and the fluorescence probe are arranged in a fluorescence PCR instrument; and (4) comparing a collected fluorescence curve CT value with a standard curve CT value after the reaction is ended, and judging the detection result. The method can be applied to the field of molecular diagnosis on biological species.

Description

technical field [0001] The invention relates to an MP gene-based real-time fluorescent PCR detection method for cucumber green mottle mosaic virus, and a combination of detection probes and primers used in the method. Background technique [0002] Cucumber green mottle mosaic virus ( Cucumber green mottle moasic virus, CGMMV ) belongs to the family Tomatobush Viridae ( Tombusviridae ) Tobacco mosaic virus ( Tobamovirus ), is one of the important viruses that seriously damage cucurbit crops, and is a typical seed-borne virus. This virus was discovered and reported for the first time on the cucumber of England in 1935, therefore named as cucumber virus 3 (CV3) and cucumber virus 4 (CV4), described by Anisworth. There are many strains of CGMMV, including the typical strains reported early in the UK and Europe ( Cucumber green mottle mosaic virus ), Cucumber Prunus mosaic strain ( Cucumber aucuba mosaic virus ), the watermelon strain reported in Japan ( Watermelon str...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
Inventor 张卫东廖力王岚梁玉英权永兵迟远丽徐淼锋
Owner 张卫东
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