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Nucleic acid extraction kit for excrement sample, preparation method and extraction method

A nucleic acid extraction reagent and fecal sample technology, applied in the field of molecular biology, can solve problems such as short storage time, and achieve the effects of long storage time, high purity, and convenience for storage and detection.

Pending Publication Date: 2022-03-01
JIANGSU COWIN BIOTECH CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a nucleic acid extraction kit, preparation method and extraction method of a stool sample. The kit is easy to operate and can be used in conjunction with automatic nucleic acid extraction equipment. The extracted and purified DNA nucleic acid has a high yield and good purity, and does not contain nucleic acid. Enzymes and PCR inhibitors can effectively solve the problem of short storage time of DNA extracted from stool samples, which is conducive to the preservation of DNA nucleic acids and subsequent PCR detection

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  • Nucleic acid extraction kit for excrement sample, preparation method and extraction method
  • Nucleic acid extraction kit for excrement sample, preparation method and extraction method
  • Nucleic acid extraction kit for excrement sample, preparation method and extraction method

Examples

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Embodiment 1

[0066] This embodiment provides a stool sample DNA nucleic acid extraction kit and preparation method. The kit includes proteinase K, initial lysis buffer, lysis-adsorption binding buffer, magnetic bead suspension, first washing solution, second washing solution and elution buffer.

[0067] In this embodiment, each reagent component of the kit is as follows:

[0068] Proteinase K is 20mg / mL;

[0069] The initial lysis buffer contains: 100 mM Tris-HCl, 10% (w / v) SDS, 8 mM EDTA, and the pH of the lysate is 8.5.

[0070] The lysis-adsorption binding buffer contains: 50mM Tris-HCl (pH6.0-8.0), 10M guanidine hydrochloride, 4mM EDTA, 1% TritonX-100, 3% Tween-20, and its pH is 7.5;

[0071] The magnetic bead suspension contains: magnetic balls with a volume fraction of 70% (the particle size of the magnetic bead is between 10-50nm), 30% nuclease-free water, and the pH of the magnetic bead suspension is 4.5;

[0072] The first rinse liquid contains: 50mM Tris-HCl, 100mM sodium chlo...

Embodiment 2

[0075] Example 2 Fecal sample DNA extraction method (manual extraction)

[0076] The feces extraction kit reagent prepared in Example 1 was used to extract the DNA nucleic acid from the stool sample.

[0077] The specific steps for manual extraction are as follows:

[0078] 1. Sample Lysis

[0079] Fresh or frozen stool sample: Take 0.2-0.3g of fresh or frozen stool sample into a 2ml clean centrifuge tube, add 600μl of primary lysis buffer, and continuously vortex for 1 minute until the stool sample is fully homogenized.

[0080] Samples preserved in feces preservation solution: Take 300 μl of feces preservation solution sample, place it in a clean 2 mL centrifuge tube, add 300 μl of primary lysis buffer, and continuously vortex for 1 minute until the stool sample is fully homogenized.

[0081] Place the above fully homogenized sample on a constant temperature mixer, incubate for 5 minutes at 70°C, 600rpm, then incubate at 80°C, 600rpm for 10 minutes, centrifuge the sample a...

Embodiment 3

[0088] Example 3 Optimizing the Automatic Extraction Program of Feces DNA Extraction Kit

[0089] Sample: Fresh stool sample

[0090] Reagent: the extraction reagent prepared according to Example 1

[0091] Instrument: CWE2100 Automatic Nucleic Acid Extractor (Kangwei Century)

[0092] The specific operation of matching CWE2100 instrument is as follows:

[0093] 1. Sample pretreatment

[0094] Fresh or frozen stool sample: Take 0.2-0.3g of fresh or frozen stool sample into a 2ml clean centrifuge tube, add 600μl of primary lysis buffer, and continuously vortex for 1 minute until the stool sample is fully homogenized.

[0095] Samples preserved in feces preservation solution: Take 300 μl of feces preservation solution sample, place it in a clean 2 mL centrifuge tube, add 300 μl of primary lysis buffer, and continuously vortex for 1 minute until the stool sample is fully homogenized.

[0096] Place the above fully homogenized sample on a constant temperature mixer, incubate f...

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Abstract

The invention provides a nucleic acid extraction kit for an excrement sample, a preparation method and an extraction method, and relates to the technical field of biology, the kit comprises protease K, a primary lysis buffer solution and a lysis binding buffer solution; the concentration range of the protease K is 10-30 mg / mL; the primary lysis buffer solution comprises Tris-HCl with the pH value of 6.0 to 8.550 to 200 mM, SDS with the concentration of 0.5 to 30 percent (w / v) and EDTA with the concentration of 1 to 10 mM, and the pH value of 8.0 to 9.0; the lysis binding buffer solution is prepared from 50 to 200mM of Tris-HCl, 1 to 15M of guanidine hydrochloride, 1 to 10mM of EDTA (Ethylene Diamine Tetraacetic Acid), 0.1 to 5 percent of TritonX-100 and 0.1 to 5 percent of Tween-20, and the pH (Potential of Hydrogen) of the lysis binding buffer solution is 7.0 to 8.0. The DNA nucleic acid extracted and purified by the kit is high in yield and good in purity, and the problem that the preservation time of the DNA extracted from the excrement sample is short is solved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a nucleic acid extraction kit, a preparation method and an extraction method of a stool sample. Background technique [0002] Stool samples or intestinal content samples are important sample sources for intestinal disease research and intestinal clinical detection. Feces or intestinal contents contain a large number of host digestive tract exfoliated cells and free nucleic acids. These exfoliated cells often contain cancerous signals. The detection of exfoliated cells and free nucleic acids in feces or intestinal contents is non-invasive, painless, and specific. It is often used in primary screening and screening of digestive tract diseases such as colorectal cancer. It can better achieve the purpose of "early detection, early diagnosis and early treatment". It is the most effective and cost-effective way to reduce colorectal cancer (colorectal cancer) ) morbidity and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806C12Q2523/308C12Q2521/537
Inventor 殷剑峰龚波王春香
Owner JIANGSU COWIN BIOTECH CO LTD
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