Gene diagnostic reagent kit and detection method of mikania micrantha wilt virus

A diagnostic kit and virus gene technology, applied in the direction of microbial-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve problems such as economic losses, achieve enhanced ecological management, avoid spreading epidemics, and broadly apply foreground effect

Active Publication Date: 2012-07-04
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus has high genetic homology with broad bean wilt virus-1 (BBWV-1) and broad bean wilt virus-2 (BBWV-2), and can infect vegetables crops, legu

Method used

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  • Gene diagnostic reagent kit and detection method of mikania micrantha wilt virus
  • Gene diagnostic reagent kit and detection method of mikania micrantha wilt virus
  • Gene diagnostic reagent kit and detection method of mikania micrantha wilt virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A genetic diagnostic kit for Mikania wilt virus, including the following components:

[0026] 1).Template extract solution I, 1 bottle, containing Trizol;

[0027] 2). Protein removal solution II, 1 bottle, filled with chloroform;

[0028] 3). RNA Precipitation Solution III, 1 bottle, filled with isopropanol;

[0029] 4). Washing solution IV, 1 bottle, filled with 75% ethanol;

[0030] 5).Eluent V, 1 bottle, filled with sterilized double distilled water;

[0031] 6). Reaction solution VI, 1 bottle, containing PCR reaction solution, including ddH 2 O, dNTP, with mg 2+ The 10×Buffer, TaqE, primer MMWVRNA1F is: 5’-TGAGGAATTGGGAAGGTC and MMWVRNA1R is: 5’-CGACTCGGCGTCATAAAC;

[0032] 7). Reaction solution VII, 1 bottle, containing PCR reaction solution, including ddH 2 O, dNTP, with mg 2+ The 10×Buffer, TaqE, primer MMWVRNA2F is: 5’-GGAATCCCTGAAACTATGC, MMWVRNA2R is: 5’-TGCTCCACCAATCACAACA;

[0033] 8). Positive control solution VIII, 1 bottle, containing plasmid DNA...

Embodiment 2

[0038] A method for detecting Mikania wilt virus using the kit of Example 1, comprising the following steps:

[0039] 1). Take the plant sample to be tested, add an appropriate amount of liquid nitrogen and grind it into powder;

[0040] 2). Transfer the ground powder to an RNase-free 2ml sterilized centrifuge tube, add 1ml template extract solution I, mix well, and let stand at room temperature for 6 minutes;

[0041] 3). Add 200 μl protein-free solution II, cap the centrifuge tube tightly, shake vigorously for 30 seconds to mix well, and let stand at room temperature for 5 minutes;

[0042] 4). Centrifuge at 12000r / min for 12min at 4°C;

[0043] 5). Take the supernatant to another RNase-free 2ml sterilized centrifuge tube, add 500μl RNA precipitation solution III solution, mix gently, and place at room temperature for 10min;

[0044] 6). Centrifuge at 12000r / min for 12min at 4°C;

[0045] 7). Discard the supernatant, add 1ml of washing solution IV to rinse, centrifuge at ...

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Abstract

The invention discloses a gene diagnostic reagent kit and a detection method of mikania micrantha wilt virus. The reagent kit comprises two pairs of specificity primers which are respectively MMWV RNA1 F: 5'-TGAGGAATTGGGAAGGTC; MMWV RNA1 R: 5'-CGACTCGGCGTCATAAAC; MMWV RNA2 F: 5'-GGAATCCCTGAAACTATGC and MMWV RNA2 R: 5'-TGCTCCACCAATCACAACA. The detection method includes the following steps: (1) extracting RNA of a plant sample to be detected; (2) adding the two primers in the RNA and respectively obtaining two cDNA after polymerase chain reaction (PCR) amplification; and (3) respectively addingtwo primers into the cDNA and obtaining two amplification products after PCR amplification and conducting electrophoresis. If a first amplification product has a bright strip belt at 329bp and simultaneously a second amplification product has a bright strip belt at 364bp, MMWV is positive, and the fact that the plant sample to be detected carries the mikania micrantha wilt virus can be proved. The reagent kit and the method can amplify relative gene segments of the sample to be detected and carrying the MMWV virus in specificity mode, and a built PCR reaction system ensures quickness, stability and accuracy of the detection result.

Description

technical field [0001] The invention relates to a diagnostic kit and a detection method for plant viruses, in particular to a genetic diagnostic kit and a detection method for Mikania micrantha wilt virus (MMWV). Background technique [0002] Mikania micrantha H.B.K. is a perennial vine belonging to the genus MikaniaWilld in the Compositae family. At present, the organisms have invaded South China, causing serious harm to the ecological environment. Mikania wilt virus belongs to the genus Fabavirus of the family Comoviridae. Mikania micrantha infection caused symptoms such as leaf wilting, top wilting, and internode dwarfing, which could seriously inhibit the growth of Mikania micrantha. The virus has high genetic homology with broad bean wilt virus-1 (BBWV-1) and broad bean wilt virus-2 (BBWV-2), and can infect vegetables crops, leguminous crops and other important economic crops, causing serious economic losses. Currently there is no effective detection method for Mika...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 王瑞龙曾任森梁笑婷辛效威宋圆圆苏贻娟骆世明
Owner SOUTH CHINA AGRI UNIV
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