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Kit for extracting fecal microbial genome by magnetic bead method and extraction method thereof

A technology of fecal microorganisms and extraction methods, which is applied to the kit and extraction field of fecal microbial genome extraction by magnetic beads method, can solve the problems of many impurities in the extracted products, low extraction efficiency, poor integrity, etc., and achieve pollution-free operation, improve quality and Purity, guaranteed activity effect

Inactive Publication Date: 2019-01-25
TIANGEN BIOTECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a kit and extraction method for extracting fecal microbial genome by magnetic bead method, which is used to solve the problems that the existing fecal microbial genome extraction products or methods take a long time, have low extraction efficiency, have many impurities in the extracted product, and have poor integrity. Problems, through innovative research on the composition of the buffer used for lysis, precipitation, rinsing and impurity removal, and precise control of the amount and time of reagents in each step, efficient, rapid, and high-quality extraction of fecal microbial genomes has been achieved

Method used

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  • Kit for extracting fecal microbial genome by magnetic bead method and extraction method thereof
  • Kit for extracting fecal microbial genome by magnetic bead method and extraction method thereof
  • Kit for extracting fecal microbial genome by magnetic bead method and extraction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The impact of embodiment 1 different lysates on the extraction of cat feces genomic DNA

[0051] 1. Cracking of microorganisms in the stool: collect 0.25g respectively in the same cat stool sample and place them in 2mL centrifuge tubes respectively, set up four experimental groups a, b, c and d, and add 0.25-0.5g to group a according to the method of the present invention Solid particles, 500-1000 μL buffer A, 50-100 μL buffer B and 10-20 μL buffer C; group b added 500-1000 μL buffer A, 50-100 μL buffer B and 10-20 μL buffer C; group c Add 500-1000 μL of buffer A and 10-20 μL of buffer C; add 50-100 μL of buffer B and 10-20 μL of buffer C to group d, fill each group with distilled water to the same volume as group a, and shake for 15 minutes to sample Mix well to obtain a cracking mixture; wherein, the solid particles added in group a are glass beads of 50 μm-1 mm;

[0052] ②Remove impurities: centrifuge the lysed mixture at 12000rpm (~13,400×g) for 1min, transfer the ...

Embodiment 2

[0058] The impact of embodiment 2 different precipitants on cat feces genomic DNA extraction

[0059] ① Lysis of microorganisms in feces: Collect 0.25g from the same cat feces sample and place them in 2mL centrifuge tubes, set up four experimental groups a, b, c, and d, and add 0.25-0.5g solids to each of the four groups of samples Particles, 500-1000 μL buffer A, 50-100 μL buffer B and 10-20 μL buffer C, respectively shake for 15 minutes until the sample is mixed to obtain a lysis mixture;

[0060] ② Remove impurities: centrifuge the lysed mixture at 12000rpm (~13,400×g) for 1min,

[0061] Group a added buffer D with 1 / 5 of the supernatant volume, group b added 100-200mM ammonium aluminum sulfate with 1 / 5 of the supernatant volume, group c added 3-5M potassium acetate with 1 / 5 of the supernatant volume, and mixed Place on ice for 8 minutes, centrifuge at 12000rpm for 3 minutes, transfer the supernatant to a new 2mL centrifuge tube, transfer the supernatant directly to a new ...

Embodiment 3

[0067] Example 3 Different methods and kit products are extracted from cat feces genome

[0068]Set up two experimental groups a and b, collect 0.3g from the same cat feces sample and place them in 2mL centrifuge tubes respectively, and use the Tiangen column method fecal genome extraction kit (DP328) for group a to carry out the steps according to its instructions. For the extraction of genomic DNA, group b adopts the kit and method of the present invention to extract, and the specific operations are as follows:

[0069] First, add 500 μL of buffer A, 100 μL of buffer B, 20 μL of buffer C and 0.25 g of glass beads to the sample in the centrifuge tube, vortex for 2 minutes until the sample is mixed, and then shake and mix for 15 minutes to obtain a lysis mixture.

[0070] Centrifuge the lysis mixture at 12,000rpm (~13,400×g) for 1min, transfer the supernatant (about 500μL) to a new 2mL centrifuge tube, add 200μL buffer D and mix well to remove impurities, vortex for 5s and pla...

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Abstract

The invention discloses a kit for extracting fecal microbial genome by magnetic bead method and an extraction method thereof, belongs to the field of biotechnology; the technical scheme adopted is; akit for extracting fecal microbial genome by magnetic bead method include lysis fluid, impurity removal reagent, genomic adsorbent and eluent, in buff a, B and C combine lysis microorganisms, the humic acid and other impurities are removed by the unique precipitating solution, then adsorbing genomic DNA with magnetic beads, further removing foreign proteins and salt ions with special buffer G andbuffer H, and finally eluting the purified genomic DNA; a method for extract genomic DNA of faeces microorganism includes such step as extracting genomic DNA of faeces microorganism with high efficiency and sufficient quantity, extracting genomic DNA of faeces microorganism with high quality, extracting genomic DNA of faeces microorganism with simple procedure, high speed, safety and no pollution,shortening extracting time, and can be used for manual operation and automatic platform, and is widely suitable for extracting animal faeces genomic DNA of faeces microorganism with high speed, efficiency and economy for customers, and has wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit and an extraction method for extracting fecal microbial genomes by a magnetic bead method. Background technique [0002] There are complex, large and relatively stable microbial flora in the intestinal tract of humans and animals. The number of microorganisms in the intestinal tract is 10 times that of the body cells, and the proportion of microorganisms in the dry weight of feces is as high as 60%. The number and types of microorganisms in the intestine are closely related to the health and disease of the body, so the study of the distribution of microorganisms in the intestine has become one of the hotspots. In 2010, the European Union funded the "Human Intestinal Metagenome Project" to study the microbial flora in the human intestinal tract, aiming to study the correlation between the number and types of microorganisms in the intestinal tract and diseases such as diabetes an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2527/125
Inventor 王金辉
Owner TIANGEN BIOTECH BEIJING
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