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RPA (recombinase polymerase amplification) method for diagnosis and pathogen detection of blood flesh disease of watermelons

A pathogen and watermelon technology, which is applied in the RPA field of watermelon blood-flesh disease diagnosis and pathogen detection, to achieve the effects of shortening identification time, saving biochemical reagents, and reducing costs

Pending Publication Date: 2019-03-15
SHENYANG AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Although this method basically meets the requirements for the detection of cucumber green mottle mosaic virus, there is still a lot of room for improvement in terms of sensitivity.

Method used

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  • RPA (recombinase polymerase amplification) method for diagnosis and pathogen detection of blood flesh disease of watermelons
  • RPA (recombinase polymerase amplification) method for diagnosis and pathogen detection of blood flesh disease of watermelons
  • RPA (recombinase polymerase amplification) method for diagnosis and pathogen detection of blood flesh disease of watermelons

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Experimental program
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Effect test

Embodiment

[0026] 1. CGMMV gene-specific primers

[0027] According to the genome sequence of CGMMV-lnxg isolate (KY040049.1), according to the design principles of RPA primers, the specific RPA primer pair CGMMV-F / CGMMV-R for detecting CGMMV was designed using software and manual correction. The primer sequences are shown in Table 1.

[0028] 2. Rapid extraction of total RNA from watermelon samples

[0029] Put 0.1 g of sample material into a cleaned and sterilized mortar and grind with liquid nitrogen, then add 1 mL of Trizol Reagent and mix well. Transfer the above homogenate sample to a 2ml RNase-free centrifuge tube, cap it, shake it vigorously and mix it evenly, and place it at room temperature for 5 minutes; add 0.2mL chloroform, mix it upside down for 15 seconds, and place it at room temperature for 15 minutes; 4°C, 12,000rpm, Centrifuge for 20min, transfer the upper colorless aqueous phase to a new 1.5ml RNase-free centrifuge tube; add an equal volume of isopropanol (about 400-...

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Abstract

The invention discloses a RPA (recombinase polymerase amplification) method for diagnosis and pathogen detection of blood flesh disease of watermelons. An efficient and economic technical system for rapidly detecting a CGMMV (cucumber green mottle mosaic virus) is established with a technological means of modern molecular biology study, the RPA rapid detection method is provided, and the method comprises steps as follows: cDNA of a sample with the virus is taken as a template in a RPA reaction, a pair of detection primers are added, a reaction is performed for 20-30 min at constant temperaturebeing 37-40 DEG C in a water bath kettle, sensitivity reaches 10<-6> and is higher, the CGMMV is detected rapidly and accurately, meanwhile, identification time is shortened, biochemical reagents aresaved, cost is reduced, and theoretical basis and technical support are provided for monitoring, forecasting and control of the CGMMV as a quarantine disease.

Description

technical field [0001] The technical field of plant quarantine of the present invention relates in particular to an RPA method for the diagnosis of watermelon blood flesh disease and the detection of pathogens. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, is an important quarantine virus on cucurbit crops worldwide, seriously threatening watermelon, muskmelon, Production of Cucurbitaceae crops such as cucumbers. CGMMV can be transmitted by seeds, pollen, soil, water and insect vectors as well as mechanical contact. The leaves of CGMMV-infected plants will have obvious mosaic symptoms, growth retardation, and fruit deformity, which will eventually lead to a decrease in yield, resulting in huge economic losses. [0003] CGMMV has a variety of transmission methods, but the main ones are infected seeds and mechanical transmission. (1) Seed transmission: CGMMV is a typical seed-borne virus. The virus is mainly tra...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2563/107C12Q2531/119
Inventor 吴元华焦裕冰夏子豪蒋均匀
Owner SHENYANG AGRI UNIV
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