阅读说明：本技术 一种dna甲基化检测粪便采样装置、保存液及其方法 (Excrement sampling device, preservation solution and method for DNA methylation detection ) 是由 钟玙沄 于 2020-07-03 设计创作，主要内容包括：本发明公开了一种DNA甲基化检测粪便采样装置、保存液及其方法。本发明中的DNA甲基化检测粪便采样装置包括：双向贯通的筒体；筒内贯穿设置有顶杆；顶杆位于筒内的一端固定连接有活塞；活塞与筒体内侧壁滑动连接。该粪便采集装置及本发明中的保存液克服了目前市场上该领域产品的空白,既可以有效保存DNA以及其中甲基化特征,又可以提高用于粪便DNA甲基化检测的样本采集的可靠性及有效性,进一步大规模进行肠癌粪便DNA甲基化筛查提供帮助。(The invention discloses a DNA methylation detection excrement sampling device, preservation solution and a method thereof. The DNA methylation detection feces sampling device comprises: a cylinder body which is communicated in two directions; a mandril penetrates through the cylinder; one end of the ejector rod positioned in the cylinder is fixedly connected with a piston; the piston is connected with the inner side wall of the cylinder in a sliding manner. The excrement collecting device and the preservation solution overcome the blank of products in the field in the current market, can effectively preserve DNA and methylation characteristics in the DNA, can improve the reliability and effectiveness of sample collection for excrement DNA methylation detection, and further provide help for large-scale intestinal cancer excrement DNA methylation screening.)

1. A stool preservation solution, characterized in that, the components of the stool preservation solution include: trihydroxymethyl aminomethane hydrochloric acid buffer solution, sodium chloride, ammonium sulfate and ethylenediamine tetraacetic acid.

2. The stool preservation solution according to claim 1, wherein the concentration of the components of the stool preservation solution is: 10-1000mM of trihydroxymethyl aminomethane hydrochloric acid buffer solution, 1-10mM of sodium chloride, 1-100mM of ammonium sulfate and 1-100mM of ethylenediamine tetraacetic acid.

3. The stool preservation solution according to claim 2, wherein the concentration of the components of the stool preservation solution is: 50-150mM of trihydroxymethyl aminomethane hydrochloric acid buffer solution, 1-10mM of sodium chloride, 1-100mM of ammonium sulfate and 1-100mM of ethylenediamine tetraacetic acid.

4. A DNA methylation detection fecal sampling device, the fecal sampling device comprising:

a cylinder body which is communicated in two directions;

a mandril penetrates through the cylinder;

one end of the ejector rod positioned in the cylinder is fixedly connected with a piston;

the push rod and the inner wall of the cylinder are provided with a clamping groove for limiting the moving range of the piston;

the piston is connected with the inner side wall of the cylinder in a sliding manner.

5. The fecal sampling device of claim 4 wherein the end of the ejector pin outside the barrel is fixedly connected with a press block.

6. A method for collecting DNA methylation detection feces, which is characterized by comprising the following steps:

inserting a fecal sampling device according to any of claims 4-5 into a fecal sample, allowing the fecal sample to enter the device;

transferring the fecal sample within the device to a fecal preservation solution according to any of claims 1-3;

DNA methylation detection was performed on stool samples in the stool preservation solution.

7. The fecal sampling device of claim 6 wherein the volume ratio of fecal preservative fluid to fecal sample is (3-7): (1-3).

8. The faecal sampling device according to claim 6, wherein said faecal sampling device contains a mass of faecal sample from 1 to 20 g.

9. Use of a stool preservation solution according to any one of claims 1-3 for the detection of DNA methylation in stool.

10. Use of a stool preservation solution according to any one of claims 1-3 for the preparation of a colorectal cancer stool DNA diagnostic and screening test product.

Technical Field

The invention relates to the field of fecal molecular detection, in particular to a fecal sampling device, a preservation solution and a method for DNA methylation detection.

Background

Colorectal cancer is the 5 th malignant tumor with morbidity and mortality in China. The early diagnosis and screening of the colorectal cancer can effectively reduce the mortality rate of malignant tumors. The fecal DNA methylation detection is a non-invasive, high-sensitivity and high-specificity detection means, is obviously superior to the current enteroscopy and fecal occult blood test, and the effectiveness of the methylation detection is written in the American colorectal cancer screening guideline and the consensus of Chinese colorectal cancer screening experts.

The accuracy of fecal DNA methylation detection is affected to some extent by the collection of fecal samples and the preservation of DNA methylation characteristics therein. The conventional method for preserving the excrement is liquid nitrogen quick freezing, and the excrement sample preserved at the temperature of between 20 ℃ below zero and 80 ℃ below zero undoubtedly increases the operability of the test and cannot be suitable for various collection environments. And the common excrement preservation solution has poor stability, and even if the common excrement preservation solution can be used for normal-temperature preservation, the preservation condition of the DNA methylation characteristics in the sample cannot be guaranteed.

Moreover, there are no patents and technologies on the collection and preservation of fecal samples for DNA methylation detection. Conventional excrement storage liquid and a collector both have higher detection failure rate, and can not conveniently and quickly measure and collect excrement samples.

Because the proportion of human-derived DNA in the feces is small, the quantity of feces to be collected is obviously increased relative to the current fecal occult blood test. The excrement and urine collection system who sees something common on the market now is mainly used for excrement occult blood test, and main problem lies in: 1) the collection amount is small; 2) the stool consistency causes different dosage.

Therefore, in order to solve the above-mentioned drawbacks, a rapid and efficient fecal sampling device for DNA methylation detection, a preservation solution and a method thereof are required.

Disclosure of Invention

One of the purposes of the invention is to provide a feces preservation solution for DNA methylation detection;

another object of the present invention is to provide a fecal sampling device for DNA methylation detection;

another objective of the invention is to provide a method for collecting DNA methylation detection feces;

the invention also aims to provide the application of the excrement storage solution in the excrement DNA methylation detection;

the invention also aims to provide application of the excrement storage liquid in preparing a colorectal cancer excrement DNA diagnosis and screening test product.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a feces preservation solution for DNA methylation detection comprises the following components: tris (hydroxymethyl) aminomethane hydrochloride buffer (Tris-HCl), sodium chloride (NaCl), ammonium sulfate, and ethylenediaminetetraacetic acid (EDTA).

Further, the concentration of the components of the feces preservation solution is as follows: 10-1000mM of trihydroxymethyl aminomethane hydrochloric acid buffer solution, 1-10mM of sodium chloride, 1-100mM of ammonium sulfate and 1-100mM of ethylenediamine tetraacetic acid.

Further, the concentration of the components of the feces preservation solution is as follows: 50-150mM of trihydroxymethyl aminomethane hydrochloric acid buffer solution, 1-10mM of sodium chloride, 1-100mM of ammonium sulfate and 1-100mM of ethylenediamine tetraacetic acid.

In a second aspect of the present invention, there is provided:

a DNA methylation detection fecal sampling device, the fecal sampling device comprising:

a cylinder body which is communicated in two directions;

a mandril penetrates through the cylinder;

one end of the ejector rod positioned in the cylinder is fixedly connected with a piston;

the push rod and the inner wall of the cylinder are provided with a clamping groove for limiting the moving range of the piston;

the piston is connected with the inner side wall of the cylinder in a sliding manner.

Furthermore, one end of the ejector rod, which is positioned outside the cylinder, is fixedly connected with a pressing block.

The inventor finds that the concentration and purity of the excrement DNA extracted by the collecting device are obviously superior to those of the collecting device on the market at present.

In a third aspect of the present invention, there is provided:

a method for collecting DNA methylation detection feces comprises the following steps:

inserting the excrement sampling device into an excrement sample, and enabling the excrement sample to enter the device;

transferring the fecal sample in the device to the fecal preservation solution;

DNA methylation detection was performed on stool samples in the stool preservation solution.

Further, the volume ratio of the feces preservation solution to the feces sample is (3-7): (1-3).

Further, the mass of the stool sample contained in the stool sampling device is 1-20 g.

In a fourth aspect of the present invention, there is provided:

the application of the feces preservation solution in the feces DNA methylation detection.

DNA abnormal methylation is an early event of malignant tumor occurrence and development, and is one of detection indexes of malignant tumor.

In a fifth aspect of the present invention, there is provided:

the excrement storage liquid is applied to preparing colorectal cancer diagnosis and screening test products.

The detection of DNA methylation in the excrement is more accurate than the excrement occult blood test which is mainly used clinically, and particularly on the detection of precancerous lesions such as adenoma and the like. Therefore, fecal DNA methylation detection is an important tool for national intestinal cancer screening.

The invention has the beneficial effects that:

1. the excrement collecting device and the preservation solution for excrement DNA methylation detection overcome the blank of products in the field in the market at present, and the excrement collecting device and the preservation solution for excrement DNA methylation detection are created and provided.

2. The preservation solution for fecal DNA methylation detection can effectively preserve DNA and methylation characteristics thereof, can improve the reliability and effectiveness of sample collection for fecal DNA methylation detection, and can improve the preservation of DNA samples for detecting DNA methylation, thereby providing help for further large-scale intestinal cancer fecal DNA methylation screening.

Drawings

FIG. 1 is a schematic view of a DNA methylation detection fecal sampling apparatus of the present invention;

FIG. 2 is a schematic illustration of a fecal sampling device of the present invention inserted into a fecal sample;

FIG. 3 is a schematic illustration of transfer of a fecal sample within a device of the present invention into a fecal preservation solution;

FIG. 4 shows the result of the methylation fluorescent quantitative PCR assay;

fig. 5 shows the results of the time performance test of the stool specimen preserved by the preservation solution of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.