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Method for rapidly extracting fecal bacterial genome DNA and virus RNA through paramagnetic particle method

An extraction method and genome technology, applied in the fields of medicine and molecular biology, can solve the problems of cumbersome steps, long time and high price, and achieve the effects of large extraction amount, stable quality and easy operation.

Inactive Publication Date: 2016-09-21
杭州卓立纳米科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to aim at the defect of above-mentioned prior art, provide a kind of magnetic bead method fecal bacterial genome DNA / viral RNA rapid extraction method, overcome the traditional method step in the prior art is loaded down with trivial details, time-consuming and laborious, the kit method is expensive and extracts The deficiencies of the stool DNA / RNA output are not high, the purity is low, the time is long, and the effect is not ideal

Method used

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  • Method for rapidly extracting fecal bacterial genome DNA and virus RNA through paramagnetic particle method

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Experimental program
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Effect test

Embodiment 1

[0023] The steps of the extraction method that the stool bacterial genome DNA of the present embodiment extracts are:

[0024] 1. Pretreatment of feces: Weigh 200mg~500mg of human or animal feces into a 2mL centrifuge tube, add 300ul~3mL buffer A, after fully crushing, take 100ul~1mL of the supernatant into a new centrifuge tube for subsequent experiments.

[0025] 2. Lysis of microorganisms in feces: Add 200ul~2mL buffer B and 5~50ul buffer C to the spare centrifuge tube mentioned above, vortex and oscillate to mix evenly, and heat in a water bath at 37°C~95°C for 10 minutes to 1 hour.

[0026] 3. Impurity removal and DNA / RNA adsorption: Add 10-100ul buffer D and 100ul-1mL buffer E after lysis, vortex to mix well, and let stand for 1-10min. Put the centrifuge tube into the magnetic stand for magnetic separation, and remove the liquid in the centrifuge tube.

[0027] 4. Removal of residual impurities: Add 100ul ~ 1000ul buffer F and buffer G respectively, vortex and shake for...

Embodiment 2

[0038] The steps of the extraction method that the stool bacterial genome DNA of the present embodiment extracts are:

[0039] 1. Pre-treatment of feces: Weigh 200mg of human or animal feces into a 2mL centrifuge tube, add 500ul~1mL buffer A, after fully crushing, take 200ul~500ul of the supernatant into a new centrifuge tube for subsequent experiments.

[0040] 2. Lysis of microorganisms in feces: add 200-500ul buffer B and 20ul buffer C to the spare centrifuge tube mentioned above, and vortex to mix.

[0041] Evenly, heat in a water bath at 37°C to 95°C for 30 minutes to 1 hour. After the lysis is completed, take 500ul ~ 1mL of the supernatant and put it in a new centrifuge tube.

[0042] 3. Adsorption of DNA: Add 20-50ul of buffer D and 300-500ul of buffer E to the above centrifuge tube, vortex and oscillate to mix well, and let stand for 5min. Put the centrifuge tube into the magnetic stand for magnetic separation, and remove the liquid in the centrifuge tube.

[0043]4...

Embodiment 3

[0049] The steps of the extraction method of the stool viral RNA of the present embodiment are:

[0050] 1. Pretreatment of feces: Weigh 200mg of human or animal feces into a 2mL clean centrifuge tube, add 500ul~1mL bufferA, after fully crushing, take 200ul~5ul of the supernatant into a new centrifuge tube for subsequent experiments.

[0051] 2. Lysis of microorganisms in feces: Add 300-500ul buffer B and 20ul buffer C to the above spare centrifuge tube, vortex and oscillate to mix evenly, and heat in a water bath at 37°C-95°C for 5-30 minutes.

[0052] 3. RNA adsorption: Add 20-50ul of buffer D and 300-500mL of buffer E after lysis, vortex to mix well, and let stand for 5min. Put the centrifuge tube into the magnetic stand for magnetic separation, and remove the liquid in the centrifuge tube.

[0053] 4. Removal of residual impurities: add 100ul ~ 1000ul buffer F and buffer G in turn, vortex and shake for 3 ~ 30 seconds, mix thoroughly, then perform magnetic separation, and ...

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Abstract

The invention discloses a method for rapidly extracting fecal bacterial genome DNA and virus RNA through a paramagnetic particle method. The method comprises the steps that 1, faeces is taken and put into a centrifuge tube, a buffer A is added, crushing and standing are conducted, and supernatant is taken and put into a new centrifuge tube; 2, a buffer B and a buffer C are added into the supernatant in sequence, and water-bath heating is conducted; 3, adsorption of DAN and RNA is conducted, wherein after decomposition is completed, a buffer D and a buffer E are added in sequence, vortex oscillation, standing and magnetic separation are conducted, and solid substances are left; 4, a buffer F and a buffer G are added into a centrifuge tube with left solid substances in sequence, vortex oscillation and magnetic separation are conducted; 5, a buffer H is added into the centrifuge tube subjected to treatment in the fourth step, vortex oscillation and water-bath heating are conducted, vortex oscillation is conducted again, cooling and magnetic separation are conducted, and the supernatant is moved to a new centrifuge tube for use.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and medicine, in particular to a method for rapidly extracting feces bacterial genomic DNA / viral RNA by a magnetic bead method. Background technique [0002] Intestinal flora is an important part of the body. Changes in the number and types of the flora structure are closely related to the health and disease of the body. The health status of humans and animals can be determined by the interaction of external environmental factors with the body itself and the microbial flora in the body. Therefore, it is of great significance to study the interaction between the host and the flora in vivo. [0003] Feces are similar to intestinal contents and contain a large number of microorganisms, plants, and intestinal exfoliated cells. Taking fecal microorganisms as the research object is conducive to better research on intestinal microorganisms, and feces sampling is convenient and non-invasive. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2563/143C12Q2563/149
Inventor 杨磊
Owner 杭州卓立纳米科技有限公司
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