Unlock instant, AI-driven research and patent intelligence for your innovation.

Molecular standard sample for bull's eye rot bacteria on apples and preparation method thereof

A technology of molecular standard samples and fruit rot pathogens, applied in the field of plant quarantine technology research, can solve the problems of high price, achieve good stability, excellent physical and chemical properties, and improve accuracy

Inactive Publication Date: 2013-01-09
刘冉 +3
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the detection of plant pathogens at home and abroad, most of the strains purchased from the American Type Culture Collection (ATCC) are used as positive controls, which are very expensive and almost impossible to purchase in China. However, there are few relevant research reports on molecular DNA standard materials for molecular detection of plant pathogens, so It is of great practical significance to conduct comprehensive and in-depth research on the preparation technology and stability assurance technology of DNA standard samples for the detection of plant pathogenic bacteria, actively carry out the development of molecular DNA standard samples for the detection of plant quarantine pathogens in my country, and fill in the gaps in this measurement field.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Molecular standard sample for bull's eye rot bacteria on apples and preparation method thereof
  • Molecular standard sample for bull's eye rot bacteria on apples and preparation method thereof
  • Molecular standard sample for bull's eye rot bacteria on apples and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Apple oxeye fruit rot pathogen molecular standard sample and its preparation method are carried out according to the following steps:

[0045] (1) Potato sucrose (PD) liquid culture medium was used for the strain of oxeye fruit rot of apples, and cultured at 20-22°C for 5-7 days.

[0046] (2) Extract the DNA of oxeye fruit rot of apples, and use the CTAB method to extract the DNA. The specific method is:

[0047] ①Take 0.1 g of dry mycelium and place it in a pre-cooled mortar, quickly grind it into a powder in liquid nitrogen, and quickly transfer the powder into a 1.5 ml centrifuge tube (do multiple tubes at the same time);

[0048] ② Add 800 μl of CTAB extraction buffer preheated at 65°C to the centrifuge tube containing dry mycelium powder in ①, vortex and oscillate to mix thoroughly, bathe in 65°C water for 50 min, and gently invert and mix once every 10 min ;

[0049] ③ Cool to room temperature, then add 800 μl of chloroform: isoamyl alcohol mixture (volume ratio...

Embodiment 2

[0069] Carry out DNA integrity, uniformity, stability test to the standard sample that embodiment 1 makes:

[0070] 1. DNA Quality and Integrity Testing

[0071] The prepared DNA was taken for gel electrophoresis, purity and concentration detection.

[0072] (1) DNA integrity detection: direct method - check by direct electrophoresis of the extracted DNA, 120V, 1.5% agarose gel electrophoresis, observe the integrity of the bands, the test results are shown in Figure 1, the bands are complete, no diffuse, It shows that the integrity of the DNA band is good.

[0073] (2) Detection of DNA concentration and purity: UV spectrophotometer method - absorb 1 μl of DNA and use a spectrophotometer to measure the absorbance at 260nm and 280nm, and then according to the OD 260 / OD 280 value to judge the DNA purity, and according to the OD 260 Calculate its concentration. Calculated according to the following formula:

[0074] DNA concentration (ng / μl) = OD 260 ×50

[...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a molecular standard sample for bull's eye rot bacteria on apples and a preparation method thereof, and belongs to the field of the research on plant quarantine technologies. The molecular standard sample for the bull's eye rot bacteria on the apples is prepared from high-purity deoxyribonucleic acid (DNA) extracted from bacterial strains of the bull's eye rot bacteria on the apples. The standard sample has the following physical and chemical properties: (1) the standard sample is pure white powder in shape; (2) the content in each tube is 5+ / -0.5mu g; (3) the purity of DNA is that OD260 / 280 is equal to 1.8 to 2; and (4) the standard sample is easily soluble in water or a TE (Tris-EDTA) buffer solution. The molecular standard sample for the bull's eye rot bacteria on the apples is obtained by the following steps of: culturing the bacterial strains, which are taken raw materials, of the bull's eye rot bacteria on the apples in a large quantity under specific conditions, extracting the high-purity DNA, split-charging, and freeze-drying to obtain a molecular standard of the molecular standard sample for the bull's eye rot bacteria on the apples. The standard sample has excellent physical and chemical properties and can be detected by using a specific primer by a polymerase chain reaction (PCR) method, and the sensitivity is 4 to 10 times. Through detectiontests, the DNA of the standard sample has high integrity, uniformity and stability; and the standard used as a positive reference substance can be used for import and export detection so as to improve the accuracy of detection results.

Description

technical field [0001] The invention relates to a molecular standard sample of apple oxeye fruit rot fungus, and also relates to a preparation method thereof, which belongs to the field of plant quarantine technology research. Background technique [0002] There are many reports on reference materials of animal viruses and food microorganisms at home and abroad, but the research on reference materials of plant pathogenic bacteria has just started, and many problems need to be solved urgently. In the detection of plant pathogens at home and abroad, most of the strains purchased from the American Type Culture Collection (ATCC) are used as positive controls. The price is very expensive, and it is almost impossible to buy them in China. However, there are few relevant research reports on the molecular DNA standard materials for the molecular detection of plant pathogens. Therefore, It is of great practical significance to conduct comprehensive and in-depth research on the prepara...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 刘冉曹际娟王有福李鑫
Owner 刘冉