Nucleic acid extraction lysate for cast-off cells in human feces and preparation method and application of nucleic acid extraction lysate

A technology of cell nucleic acid and lysate, which is applied in the field of nucleic acid extraction and lysate from exfoliated cells in human feces, which can solve the difficulty of nucleic acid purification and enrichment, difficulty in obtaining high-purity and high-yield nucleic acid, and difficulty in meeting the dosage of tumors and other problems, to achieve rapid and efficient lysis, reduce toxic reagents, and be suitable for large-scale application

Active Publication Date: 2018-04-20
SHANGHAI TELLGEN LIFE SCI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the composition of feces is very complex, and there are many impurities in the sample, such as food residues, bile salts, bilirubin, humus, pigments, etc., which make the purification and enrichment of nucleic acids in fecal samples more difficult.
[0005] It is difficult to obtain high-purity and high-yield nucleic acid with conventional nucleic acid lysis solution for stool samples, and it is difficult to meet the amount required for subsequent related tumor detection

Method used

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  • Nucleic acid extraction lysate for cast-off cells in human feces and preparation method and application of nucleic acid extraction lysate
  • Nucleic acid extraction lysate for cast-off cells in human feces and preparation method and application of nucleic acid extraction lysate
  • Nucleic acid extraction lysate for cast-off cells in human feces and preparation method and application of nucleic acid extraction lysate

Examples

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preparation example Construction

[0067] The present invention also provides the preparation method of the above-mentioned exfoliated cell nucleic acid extraction lysate in human feces, comprising the following steps:

[0068] (1) adding the metal ion chelating agent, the buffer substance of the buffer solution and the guanidinium salt in deionized water, and dissolving and mixing the components to obtain a mixture;

[0069] (2) Adjust the pH of the mixture to 7.5-8.0.

[0070] Preferably, in the case that the lysate for extracting nucleic acid from exfoliated cells in human feces further includes 1% to 20% by volume of a surfactant, the preparation method of the lysate for extracting nucleic acid from exfoliated cells in human feces further includes :

[0071] (3) Add the surfactant into the mixture and mix well.

[0072] Thus, the lysate for extracting nucleic acid from exfoliated cells in human feces according to the present invention can be efficiently prepared, and the cost is low, and it can be effecti...

Embodiment 1

[0119] Example 1 Comparison of different guanidinium salts in the lysate for the extraction of nucleic acid from exfoliated cells in human feces

[0120] Different chaotropic agent guanidine salts (guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate) were added to 100mM Tris containing a certain concentration of metal ion chelating agent 20mM disodium oxalate tetraacetate and pH8.0 -HCl buffer solution, and set a blank control (100mM Tris-HCl buffer solution containing 20mM disodium oxalate tetraacetate and pH8.0 without guanidine salt) to form nucleic acid extraction lysates of different compositions, according to the stool nucleic acid extraction process The operation was carried out to extract DNA, and three samples were processed in parallel in each group of experiments, and then fluorescent quantitative PCR detection was performed in the amplification system containing primer probes designed for human globin (β-globin) related genes. The primer sequen...

Embodiment 2

[0126] Example 2 Compare different concentrations of guanidinium isothiocyanate in the lysate to the human exfoliated cell nucleic acid extraction effect in the feces

[0127] For further screening more suitable guanidinium isothiocyanate concentration, carried out the preparation of different concentrations of guanidine isothiocyanate by table 3, all components are all added in the metal ion chelating agent disodium oxalate tetraacetate of 20mM and 100mM Tris-HCl buffer solution at pH 8.0. Refer to Example 1 for the remaining steps.

[0128] Table 3 compares the effect of different concentrations of guanidine isothiocyanate in the lysate on the nucleic acid extraction of exfoliated cells in human feces

[0129]

[0130] The above results show that increasing the guanidinium isothiocyanate has an effect on the yield of nucleic acid extraction. Guanidine isothiocyanate can effectively improve the nucleic acid extraction effect of human exfoliated cells in the feces in the c...

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Abstract

The invention provides a nucleic acid extraction lysate for cast-off cells in human feces. The nucleic acid extraction lysate comprises 1-8 mol/L guanidinium, a 1-20 mM metal ion chelating agent and a100 mM Tris-HCl buffer solution with the pH of 7.5-8.0, wherein guanidinium is selected from at least one of guanidine hydrochloride, guanidinium isothiocyanate and guanidine thiocyanate; the metal ion chelating agent is selected from at least one edetic acid and water soluble salt of edetic acid. Preferably, the nucleic acid extraction lysate further comprises a surfactant with the volume of 1-20%, the surfactant is a non-ionic detergent, and the non-ionic detergent is selected from at least one of Tween-20, Tween-80, Brij-35, NP40 and Triton-100. The invention further provides a related preparation method and application. The nucleic acid extraction lysate can quickly and efficiently realize cell lysis, fully releases nucleic acid, meets the consumption demand for follow-up nucleic aciddetection and is suitable for large-scale popularization.

Description

technical field [0001] The present invention relates to the technical field of nucleic acid extraction, in particular to the technical field of nucleic acid extraction lysate, in particular to a nucleic acid extraction lysate from exfoliated cells in human feces, its preparation method and application. Background technique [0002] As a common clinical sample, stool contains a large amount of nucleic acid information, which can be used for the detection of related tumors, and the most common use is for the diagnosis of colorectal cancer. [0003] A large number of studies have shown that feces contain not only exfoliated normal colorectal epithelial cells and free DNA, but also a large number of cancer cells and free cancer DNA shed from colorectal cancer; and because the intestinal tract is an alkaline environment, it is conducive to DNA Preservation, so that the quality of DNA extracted from feces is better. Therefore, fecal DNA is the best specimen for molecular detectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806C12Q2563/143C12Q2563/149
Inventor 左红伟李志甲朱凤
Owner SHANGHAI TELLGEN LIFE SCI CO LTD
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