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

Method for extracting surface bacterium genome of aged tobacco leaves

A genome and bacterial technology, applied in the field of microbiology, can solve problems such as unstable DNA quality, poor effect, and interruption of experiments, and achieve the effects of avoiding adverse effects, wide application range, and increasing acquisition rate

Inactive Publication Date: 2016-09-21
CHINA TOBACCO YUNNAN IND
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the commercial DNA extraction kits currently on the market can obtain high-quality genomic DNA, they are only for purely cultured microorganisms, and the kits for directly extracting microbial genomic DNA from environmental samples are less effective, and non-microbial substances will clog DNA adsorption column, and the quality of extracted DNA is unstable
The method of manually extracting the genome of the sample is inefficient and easily leads to the loss of the genome
When directly using the kit to extract the genomic DNA of bacteria on the surface of tobacco leaves, small tobacco leaf fragments and dust will block the DNA adsorption column, affecting the efficiency and even causing the interruption of the experiment. After the flue-cured tobacco leaves are soaked, some oily substances will overflow. Viscous flocs will form, which can also clog the adsorption column

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
  • Method for extracting surface bacterium genome of aged tobacco leaves
  • Method for extracting surface bacterium genome of aged tobacco leaves
  • Method for extracting surface bacterium genome of aged tobacco leaves

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Utilizing the CTAB / NaCl method to extract the genome genome of bacteria on the surface of tobacco leaves of K326 flue-cured tobacco during aging, including the following steps:

[0037] (1) Preparation of reagents:

[0038] Sample pretreatment reagents include 1×TE buffer solution with pH=8.0, 20mg / mL lysozyme, 20% SDS by mass volume ratio, 5mol / L sodium chloride solution, CTAB / NaCl solution, and 20mg / ml protease K solution, RNaseA solution of 20 μg / ml, chloroform, isoamyl alcohol, Tris saturated phenol, isopropanol, ethanol with a mass percent concentration of 70%; the preparation method of the reagent is as follows:

[0039] The preparation of the 1L EDTA (pH=8.0, final concentration 0.5M): take 186.1g Na 2 EDTA·2H 2 O, adjusted to pH = 8.0 with NaOH (about 20 g), filtered ddH 2 Mix O to 1L, store at room temperature after high temperature and high pressure sterilization; preparation of 1M Tris-HCl (pH8.0): take 121.1g Tris, add about 42ml of concentr...

Embodiment 2

[0058] Example 2: Using DNA extraction kit to extract bacterial genomes on the surface of tobacco leaves in K326 variety aging

[0059] (1) Preparation of sample pretreatment reagents

[0060] Sample pretreatment reagents include pH 7.0 PBS buffer solution, and its preparation method is shown in Case 1. The DNA kit used the Simgen Soil DNA Purification Kit, and the use of the reagents used and the experimental operation were performed according to the kit’s operating instructions. (2) Pretreatment of leaf samples

[0061] Add 1000mL of pH 7.0 PBS buffer solution to each portion and shake fully for 1-2 hours. Filter and collect the soaking solution with double-layer gauze, centrifuge the filtrate (10000×g, 4°C) for 15-20 minutes, discard the supernatant, add 80 mL of pH 7.0 PBS buffer solution, fully resuspend the precipitate, and centrifuge at no more than 80 g (4 ℃) for 3 minutes, discard the precipitate, collect the liquid, then centrifuge the liquid (10000×g, 4 ℃) for 15...

Embodiment 3

[0067] Example 3: Extraction of bacterial genomes on the surface of tobacco leaves in K326 variety aging by using Trizol reagent in combination with the kit

[0068] (1) Preparation of sample pretreatment reagents

[0069] Sample pretreatment reagents include pH 7.0 PBS buffer solution, and its preparation method is shown in Case 1. The DNA kit used the Simgen soil DNA purification kit, and the trizol reagent was purchased in the market.

[0070] (2) Pretreatment of leaf samples

[0071] Add 1000mL of pH 7.0 PBS buffer solution to each portion and shake fully for 1-2 hours. Filter and collect the soaking solution with double-layer gauze, centrifuge the filtrate (10000×g, 4°C) for 15-20 minutes, discard the supernatant, add 80 mL of pH 7.0 PBS buffer solution, fully resuspend the precipitate, and centrifuge at no more than 80 g (4 ℃) for 3 minutes, discard the precipitate, collect the liquid, then centrifuge the liquid (10000×g, 4 ℃) for 15-20 minutes, discard the supernatan...

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 relates to a method for extracting a surface bacterium genome of aged tobacco leaves. The method comprises the two main steps of aged tobacco leaf surface bacterium enrichment and genome DNA extraction, wherein bacteria are enriched through soaking with a buffer solution and gradient centrifugation, and the bacterium genome DNA is extracted with a method combining a trizol reagent and a reagent kit. The trizol reagent can fully disrupt bacteria and effectively prevent nucleic acid matter from degrading; furthermore, through high-speed centrifugal treatment, pure nucleic acid matter can be obtained after layering, the extracting efficiency of DNA is improved, and then the acquisition rate of the genome can be effectively increased through treatment of the reagent kit. By means of the method, the surface bacterium genome DNA of aged tobacco leaves can be effectively and quickly extracted.

Description

technical field [0001] The invention relates to the field of microbiology, in particular to a method for extracting bacterial genome DNA from the surface of aged tobacco leaves. Background technique [0002] The fresh flue-cured tobacco leaves are collected after they are ripe for initial roasting, and then enter the aging section after threshing and redrying. The aging section can adjust and improve the quality of tobacco leaves and maintain the stability of tobacco leaf quality, which is essential in cigarette production processing stage. The surface and interior of the aged tobacco leaves are rich in microorganisms, and the aged flue-cured tobacco leaves are rich in various compounds, including sugars, proteins, nicotine, etc., which meet the nutritional conditions for the survival of microorganisms. Many substances in the tobacco leaves have affinity Water-based, can absorb water from the aging environment, and under suitable conditions, it will cause a large number of ...

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 Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2523/113C12Q2523/308
Inventor 王毅高锐宋鹏飞张光煦杨威魏云林浦绍占季秀玲
Owner CHINA TOBACCO YUNNAN IND