PCR detection kit for fruit tree main virus and using method

A detection kit and detection reagent technology, applied in the biological field, can solve the problems of reduced detection costs and shortages, etc.

Inactive Publication Date: 2007-08-15
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. The detection cost is further reduced, which is less than half of the foreign similar detection cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment one: a kind of PCR detection kit of fruit tree main virus, is made up of fruit tree virus RNA extraction reagent and PCR detection reagent, wherein,

[0045] Fruit tree virus RNA extraction reagents are: liquid nitrogen, RNA extraction buffer, phenol, chloroform, isoamyl alcohol, lithium chloride (LiCl), diethyl pyrocarbonate aqueous solution (concentration?), 3mol / L NaAc (pH5.3 ), absolute ethanol;

[0046] PCR detection reagents are: dNTPs 100μmol / L, 0.67mol / L Buffer buffer (pH8.8), RNase inhibitor (RNasin), MgCl 2 , primers, Mo-MLV RTase (reverse transcriptase), total RNA template, Taq enzyme, sterile double distilled water.

[0047] When the kit is used to detect apple chlorotic leaf spot virus, the procedure is as follows:

[0048] The first step: fruit tree virus RNA extraction

[0049]①Take 200 μl of fruit tree tissue, grind it into powder with liquid nitrogen, and quickly transfer it into a 1.5ml centrifuge tube, add 300 μl RNA extraction buffer an...

Embodiment 2

[0060] Embodiment two: a kind of PCR detection kit of fruit tree main virus, its composition is with embodiment one, when being used for detecting grape leafroll virus, operate as follows:

[0061] The first step: fruit tree virus RNA extraction, with embodiment one;

[0062] Step Two: Reverse Transcription

[0063] Take dNTPs 100μmol / L, 0.67mol / L Buffer (10x) buffer (pH8.8) 1μl, RNasin10U, MgCl 2 0.8mmol / L, GLR-Pc 0.4μmol / L, MMLV RTase 50U, total RNA 1μl (0.5-1.0μg). with DEPC ddH 2 Make up 10 μl of O, mix and incubate at 42°C for 1 h;

[0064] The third step: PCR amplification

[0065] Take dNTPs 80μmol / L, PCR Buffer 2.5μl, MgCl 2 1.0mmol / L, GLR-Pc0.45μmol / L, GLR-P h 0.50μmol / L, Taq DNA polymerase 1U, with DEPC ddH 2 O to make up 25 μl. After mixing, react at 94°C for 30s, 52°C for 1min, and 72°C for 1min. The reaction cycle is 30 times, and the last round of extension is 7min;

[0066] Step 4: Perform electrophoresis and staining on the amplified products to gen...

Embodiment 3

[0067] Embodiment three: a kind of PCR detection kit of fruit tree main virus, its composition is with embodiment one, when being used for detecting apple stem groove virus, operate as follows:

[0068] The first step: fruit tree virus RNA extraction

[0069] ①Take 300mg of fruit tree tissue, add liquid nitrogen to grind it into powder, and quickly transfer it into a 1.5ml centrifuge tube, add 600μl RNA extraction buffer and 800μl hot phenol, shake for 60 seconds, 4°C, 12000 rpm, and centrifuge for 10 minutes;

[0070] ② Take the water phase, add 2 times the volume of phenol: chloroform: isoamyl alcohol (25:24:1), mix well, and centrifuge at 12000rpm at 4°C for 10 minutes;

[0071] ③ Take the water phase, add 2.5 times the volume of LiCl, and store it at 4°C for 2 hours;

[0072] ④ The precipitate was dissolved in 250 μl of 0.1% diethyl pyrocarbonate aqueous solution, and after standing at room temperature for 1.5 hours, it was extracted twice with chloroform:isoamyl alcohol ...

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Abstract

The invention discloses a PCR check agent case of fruit tree main virus and using method in biological technical domain, which is characterized by the following: composing by RNA extracting agent and PCR checking agent; utilizing PCR know-why; basing on agent constitution of scientific research optimism; integrating; getting the product; using to PCR test of apple chlorosis leaf speckle virus, apple micropteres virus, apple mosaic virus, plum genus necrosis ring speckle virus, grape leaf folding virus and plum dwarf virus; fitting-in PCR device and high speed refrigerated centrifuge. This invention possesses merits of high sensibility, simple operation and short time, which can be adopted by inspection department, high school and scientific research institute.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the detection of main viruses of fruit trees. technical background [0002] Virus disease of fruit trees is a disease difficult to control. Once infected, it is difficult to remove under natural conditions and remains in the tree for life. Fruit trees infected with viral diseases will destroy their physiological functions, cause poor growth, decrease in yield, and deteriorate in quality, and cause long-term chronic damage to fruit trees. In severe cases, the trees will die and bring huge economic losses to production. [0003] There are many types of fruit tree viruses, and the damage is long-lasting. So far, there is no effective drug to control them. At present, the more feasible control measure is to implement quarantine to control its expansion. Therefore, the establishment of efficient and sensitive detection technology has become a prerequisite for solving basic p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 侯义龙
Owner DALIAN UNIV
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