Two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and its detecting method

A potato virus and RT-PCR technology, applied in the field of agricultural biology, can solve the problems of difficulty in ensuring the quality of qualified seedlings, lack of positive controls, and restrictions on the potential of potato virus-free seedlings to increase production.

Inactive Publication Date: 2006-11-08
CHONGQING UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the current potato virus molecular diagnostic kits that are not produced in China, and a small number of imported serological kits have poor specificity, lack of positive controls, and inability to determine the

Method used

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  • Two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and its detecting method
  • Two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and its detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1. Two-step duplex RT-PCR simultaneous detection of potato virus Y and potato leafroll virus solid-phase detection kit and detection method.

[0135] 1. Components of the solid-phase detection kit:

[0136] Sample virus RNA extraction reagent, PVY, PLRV viral RNA reverse transcription solid-phase reagent, cDNA amplification solid-phase reagent, harmless PVY, PLRV positive control, healthy plant negative control.

[0137] 2. Preparation of solid-phase kit:

[0138] 1. The detection method includes the following steps:

[0139] 1) Extracting viral RNA: preparing potato virus RNA special extract solution (0.5% Triton X-100 and containing final concentration 0.4% NaSO 3 Mixed solution with an appropriate amount of purified water without RAN enzyme at room temperature); take out the potato sample to be tested, and add 10 mg of leaves, 30 mg of leaf stems, 40 mg of stems or 60 mg of tubers of the sample potato plants into 100 μL of potato virus RNA extract Extract ...

Embodiment 2

[0147] Example 2. Two-step triple RT-PCR simultaneous detection of Potato virus X, Potato virus A and Potato S virus solid-phase detection kit and detection method.

[0148] 1. Components of the solid-phase detection kit:

[0149] Sample viral RNA extraction reagents, PVX, PVA, PVS viral RNA reverse transcription solid-phase reagents, cDNA amplification solid-phase reagents, harmless PVX, PVA, PVS positive controls, healthy plant negative controls.

[0150] 2. Preparation of solid-phase kit:

[0151] 1. The detection method includes the following steps:

[0152] 1) extract viral RNA: prepare potato virus RNA special extraction liquid (being 0.5% Triton X-100 and containing final concentration 0.4% NaSO 3 Mixed solution with an appropriate amount of purified water without RAN enzyme at room temperature); take out the potato sample to be tested, and add 5 mg of leaves, 20 mg of leaf stems, 30 mg of stems or 50 mg of tubers of the sample potato plants into 100 μL of potato viru...

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Abstract

The present invention relates to two-step synchronous double RT-PCR process of detecting Y virus and leaf roll virus of potato, synchronous triple RT-PCR process of detecting X virus, A virus and S virus of potato, and the detecting kit. The present invention can detect five main potato viruses within 4 hr in high sensitivity, high specificity, high stability and high reliability. The present invention is suitable for the early diagnosis on potato seedling and potato seeds, and is significant on the quality monitoring of potato seed and virus disease prevention.

Description

technical field [0001] The invention relates to an agricultural biotechnology, in particular to a simple and rapid solid-phase nucleic acid detection kit for synchronously amplifying multiple potato viruses, which is suitable for use in departments such as plant protection and agricultural product quality technical supervision. Background technique [0002] Potato is a high-yield crop worldwide, and my country's planting area and output rank first in the world. Potato virus disease is an important restrictive factor in potato production and the main cause of potato variety degeneration, seriously affecting the yield and quality of potato. In my country, several main viruses and viroids that harm potatoes are Potato Virus X (PVX for short), Potato Virus Y (PVY for short), Potato Leafroll Virus (PLRV for short), Potato Virus A (PVA for short), Potato Virus S (PVS for short), wherein the most serious virus is PLRV, PVY. In potato production, seve...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王中康殷幼平袁青夏玉先彭国雄曾德玉曹月青刘红光
Owner CHONGQING UNIV
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