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Method of detecting tomato yellow leaf curl virus based on RPA (recombinase polymerase amplification)

A technology for tomato yellowing, curved leaves and viruses, which is applied in the field of plant disease diagnosis, can solve the problems that are difficult to be widely used in on-site detection, high cost, and time-consuming, and achieve reliable technical support, easy operation, and prevention of spread.

Active Publication Date: 2017-10-17
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Coupled with high cost and long time-consuming, these limitations make its application mostly limited to laboratories with perfect conditions, and it is difficult to be widely used in on-site testing

Method used

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  • Method of detecting tomato yellow leaf curl virus based on RPA (recombinase polymerase amplification)
  • Method of detecting tomato yellow leaf curl virus based on RPA (recombinase polymerase amplification)
  • Method of detecting tomato yellow leaf curl virus based on RPA (recombinase polymerase amplification)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the RPA detection nucleotide primer combination screening of tomato yellow leaf curl virus

[0044] (1) Referring to the DNA-A gene sequence of tomato yellow leaf curl virus (GenBank: KM506961.1), combined with the characteristics of RPA primer design, 11 RPA primers (Table 1) were designed to form 8 pairs of primer combinations (Table 2), for screening experiments.

[0045] (2) Weigh 0.1 g of tomato sample leaves, use TaKaRa MiniBEST Plant Genomic DNA Extraction Kit to extract sample DNA, and operate according to the kit instructions.

[0046] (3) Take 1 μL of tomato sample total DNA as a template, add ddH in sequence 2 O 13 μL, 10 μmol / L forward primer 2 μL, 10 μmol / L reverse primer 2 μL, dry powder dissolving buffer (Rehydration buffer) 29.5 μL, finally add 280 mmol / L magnesium acetate 2.5 μL to start the reaction, the total volume is 50 μL, mix After homogenization, incubate at 37°C for 40min. At the same time, healthy tomato sample DNA was used as ...

Embodiment 2

[0057] Embodiment 2, the specificity analysis of the RPA detection method of tomato yellow leaf curl virus

[0058] (1) Extract DNA from tomato yellow leaf curl virus positive samples and healthy tomato samples, tomato chlorosis virus (ToCV), cucumber mocsic virus (CMV), tobacco mosaic virus (Tobacco mosaic virus) virus, TMV) positive samples of RNA. Plant genomic DNA and RNA extraction kits were used to extract.

[0059] (2) The extracted RNA was reverse-transcribed by the following method: dNTPs (2.5mmol / L) 3μL, 5×MMLVbuffer 2μL, MMLV (200U / μL) 1μL, Rnase inhibitor (40U / μL) 1μL, random hexamer primer and Oligo( 0.5 μL each of dT)18 primers, 1 μL of plant total RNA, and sterilized ddH 2 Make up to 20 μL with O, mix well, and incubate at 42°C for 1 h to obtain cDNA.

[0060] (3) Take 1 μL each of the DNA extracted in step (1) and the cDNA obtained in step (2) as a template, and add ddH in sequence 2 O 13 μL, 10 pmol / L forward primer F3 2 μL, 10 pmol / L reverse primer R3 2 μ...

Embodiment 3

[0063] Example 3, Sensitivity Analysis of the RPA Detection Method for Tomato Yellow Leaf Curl Virus

[0064] (1) The recombinant plasmid is the plasmid pMD18-T-TYF6R6 containing the TYLCV genome, which is preserved by the Plant Disease Group of the Institute of Plant Protection and Environmental Protection, Beijing Academy of Agriculture and Forestry Sciences. The recombinant plasmid is obtained by ligating the DNA fragment (sequence 4) obtained by amplifying the TYLCV positive sample with primers TYF6:CGGATGGAAATTGTGCTGA and TYR6:ACTATCTTCCTCTGCAATC to the pMD18-T plasmid. The size of the recombinant plasmid is 2692+2155=4847bp.

[0065] The plasmid concentration was determined to be 30.3ng / μL with a micro-spectrophotometer. According to the following formula, the copy number of the plasmid is calculated as 6×10 9 copy / μL, the plasmid was used as a standard sample mother solution, and stored at -20°C for future use.

[0066] Plasmid copy number calculation formula: A=(B×6...

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Abstract

The invention discloses a method of detecting tomato yellow leaf curl virus based on RPA (recombinase polymerase amplification). The invention provides a primer pair for detecting tomato yellow leaf curl virus; the primer pair includes a primer pair (a) composed of two single-stranded DNA molecules shown by sequence 1 and sequence 2 in a sequence table, and a primer (b) as functional as the primer pair (a) and composed of two single-stranded DNA molecules shown by sequences formed by subjecting the sequence 1 and sequence 2 in the sequence table to substitution and / or deletion and / or addition of one or more nucleotides. The RPA primers herein can effectively amplify target genes, the specificity reaches 100%, and sensitivity reaches 6*105 c / mu L; the primers show no cross-reaction with common tomato viruses. The method acts as an available quick detection method for screening plantlets of nurseries, and is significant to preventing spreading of tomato yellow leaf curve virus.

Description

technical field [0001] The invention belongs to the field of plant disease diagnosis and relates to a method for detecting tomato yellow leaf curl virus based on RPA. Background technique [0002] Tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) is an important pathogen that causes tomato yellow leaf curl disease. Infected tomato plants show symptoms such as curled leaves, mottle, yellowing, shortened internodes, and plant dwarfing. . my country first discovered TYLCV in Shanghai in 2006, and then the disease spread from south to north, causing serious impact on tomato production. [0003] In the comprehensive prevention and control measures of tomato yellow leaf curl virus disease, in addition to the use of anti-virus varieties and the prevention and control of transmission media, it is also very important to prevent the occurrence of the virus disease, and rapid and accurate virus detection is an important prerequisite for prevention work . [0004] ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6844C12Q2522/101C12Q2531/119C12Q2521/507
Inventor 周莹乔鑫刘梅张玮燕继晔乔广行
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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