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Method for detecting rice stripe virus in single-head laodelphax striatellus through dot-ELISA (dot-enzyme-linked immuno sorbent assay)

A technology of rice stripe virus and SBPH, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of different physiological characteristics, no homology, no commonality, etc., to save detection time and avoid crossover of samples Pollution, the effect of saving testing costs

Inactive Publication Date: 2015-01-07
YUNNAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] The patent application number CN201210423231 discloses a dot-ELISA method for rapid detection of tomato yellow leaf curl virus carried by whitefly bemisia tabaci. Begomovirus ), the virus-transmitting agent Bemisia tabaci is an insect of the genus Bemisidae, and the rice stripe virus involved in this patent is the genus Buniaceae Fibrivirus ( Tenuivirus ) virus, and its virus-transmitting agent, SBPH, belongs to the genus SBPH. They belong to different families and genera, have different hosts, have different physiological characteristics, have no homology, and have no common ground

Method used

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  • Method for detecting rice stripe virus in single-head laodelphax striatellus through dot-ELISA (dot-enzyme-linked immuno sorbent assay)

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: The detection of RSV carried by a single head of SBPH was carried out with samples of SBPH collected in the field, and the carrier rate of SBPH was calculated.

[0031]The specific steps are as follows: Collect 30 SBPHs in the field with an insect extractor and put them into a bottle prepared with rice seedlings in advance to facilitate the survival of SBPH; store the collected SBPH in a -80°C ultra-low temperature refrigerator. During detection, pick a single-headed SBPH with a sterilized toothpick, place it in a clean 1.5mL centrifuge tube filled with three 2mm small steel balls, add 150μL of 0.01 mol / L PBS buffer to each centrifuge tube, and put Oscillate and grind for 60 s in a high-throughput tissue grinder with an oscillation frequency of 60 Hz until obvious tissue disappears. Put the centrifuge tube into a centrifuge at 4°C and centrifuge at 12000rpm for 3min, and the supernatant is ready for use. Use a pencil to draw a line on the outer paper of t...

Embodiment 2

[0032] Example 2: The detection of RSV carried by a single head of SBPH was carried out with samples of SBPH raised in a greenhouse, and the virus-carrying rate of SBPH was calculated.

[0033] The specific steps are as follows: after collecting 60 SBPHs reared in the greenhouse with an insect extractor, put the SBPH in a -20°C refrigerator for 5 minutes to freeze. Pick a single head of SBPH with a sterilized toothpick, put it into a 1.5mL centrifuge tube, add 200 μL of 0.01 mol / L PBS buffer to each centrifuge tube, and mash it with a tissue grinder until the obvious tissue disappears. Put the centrifuge tube into a centrifuge at 4°C and centrifuge at 12000rpm for 5min, and the supernatant is ready for use. Use a pencil to draw a line on the outer paper of the NC membrane, so that the NC membrane is printed with grid marks, each grid size is 0.7 × 0.7 cm, cut the membrane with a size of 7 × 9 grids, and put it flat into the center of a 9 cm diameter petri dish. Take 2 μL of t...

Embodiment 3

[0034] Embodiment 3: The detection of RSV carried by a single head of SBPH was carried out with the samples of SBPH collected in the greenhouse, and the carrier rate of SBPH was calculated.

[0035] The specific steps are as follows: After collecting 79 SBPHs reared in the greenhouse with a fluke, put the SBPHs into a -20°C refrigerator for 5 minutes to freeze. Use a sterilized toothpick to pick a single head of SBPH, put it in a clean 1.5mL centrifuge tube filled with five 1mm small steel balls, add 250μL of 0.01 mol / L PBS buffer to each centrifuge tube, and place it in a shaker Oscillating and grinding for 90 s in a high-throughput tissue grinder with a frequency of 60 Hz until obvious tissue disappears. Put the centrifuge tube into a centrifuge at 4°C and centrifuge at 12000rpm for 10min, and the supernatant is ready for use. Use a pencil to draw a line on the outer paper of the NC membrane, so that the NC membrane is printed with grid marks, each grid size is 0.7 × 0.7 cm...

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Abstract

The invention provides a method for detecting rice stripe virus in single-head laodelphax striatellus through dot-ELISA (dot-enzyme-linked immuno sorbent assay), and belongs to the field of plant protection. According to the method, after laodelphax striatellus is collected and ground, and a dot-ELISA system for detecting the rice stripe virus carried by single-head mediator laodelphax striatellus is established by monoclonal antibody of the rice stripe virus. The detection method is high in sensitivity and specificity, is suitable for large-batch single-head laodelphax striatellus sample detection, does not require special instruments and equipment, has the characteristics that the result is simple and easy to determine, the detection result is convenient to store for a long time and the like, can be used for performing large-scale survey and detection on the virus carried rate of the rice stripe virus carried by field laodelphax striatellus, and provides technical support for prediction of outbreaks of the field rice stripe disease and adoption of corresponding prevention and control measures .

Description

technical field [0001] The invention relates to a method for using dot-ELISA to detect rice stripe virus in a single-headed brown planthopper, belonging to the field of plant protection. Background technique [0002] Rice stripe virus ( Rice stripe virus , RSV) is the pathogen that causes rice stripe leaf blight, which can be caused by SBPH ( Laodelphax striatellus ), white spines planthopper ( Unkanodes sapporona ), white striped planthopper ( U. albifascia ) and white striped planthopper ( Terthron albovittatum ) transmission, but mainly depends on the persistent mode of transmission of eggs to offspring by SBPH. Rice stripe blight first occurred in Kanto, Japan in 1897, and then successively occurred in Russia, North Korea, South Korea, the former Soviet Union and Ukraine. Since it first occurred in the southern part of Jiangsu in 1963 in my country, it has rapidly expanded to 18 provinces (municipalities, autonomous regions) and vast rice areas, among which outb...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 李凡张水英李月月李梅蓉谭冠林杨耀珠马庆
Owner YUNNAN AGRICULTURAL UNIVERSITY
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