Molecular standard sample of rape stem canker pathogen and its preparation method

A technology of molecular standard samples and standard samples, which is applied in the field of plant quarantine technology research, can solve problems such as high prices, and achieve the effects of good stability and improved accuracy

Inactive Publication Date: 2011-12-14
李鑫 +3
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  • Application Information

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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 pr

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  • Molecular standard sample of rape stem canker pathogen and its preparation method
  • Molecular standard sample of rape stem canker pathogen and its preparation method
  • Molecular standard sample of rape stem canker pathogen and its preparation method

Examples

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Effect test

Embodiment 1

[0044] The preparation method of the molecular standard sample of canker sore pathogen of rapeseed is carried out according to the following steps:

[0045] (1) The canker sore strain of rapeseed was cultured in potato sucrose (PD) liquid medium at 20-22°C for 5-7 days.

[0046] (2) Extract the DNA of rapeseed canker sores, and use the CTAB method to extract the DNA. The specific steps are as follows:

[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 24:1) to ...

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

[...

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Abstract

The invention belongs to the field of plant quarantine technology research. Molecular standard sample of canker canker of rapeseed. Rape canker sore molecular standard sample is prepared from high-purity DNA extracted from canker canker sore strain. The standard sample has the following physical and chemical properties: (1) shape: pure white powder; (2) each tube Content 5μg±0.5μg; (3) DNA purity: OD260/280=1.8~2; (4) Soluble in water or TE (Tris-EDTA) buffer. The rapeseed canker sore molecular standard sample of the present invention uses the rapeseed canker sore strain isolated by the laboratory as a raw material, and is cultured in large quantities under specific conditions to extract high-purity DNA, which is subpackaged and freeze-dried to obtain the rapeseed canker sore strain. Molecular standard product, with excellent physical and chemical properties, can be detected by PCR method with specific primers, and the sensitivity can reach 10-4 times. After testing, the standard product has good DNA integrity, uniformity and stability, and can be used in import and export testing as a positive reference substance to improve the accuracy of test results.

Description

technical field [0001] The invention relates to a molecular standard sample of canker sore pathogen of rapeseed, 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 pre...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/10C12R1/645
Inventor 李鑫刘冉曹际娟王有福
Owner 李鑫
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