CN110016475B

CN110016475B - Excrement DNA extraction kit

Info

Publication number: CN110016475B
Application number: CN201910388938.8A
Authority: CN
Inventors: 周杰锋
Original assignee: Ningbo Ajcore Biotechnology Co ltd
Current assignee: Ningbo Ajcore Biotechnology Co ltd
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2021-07-06
Anticipated expiration: 2039-05-10
Also published as: CN110016475A

Abstract

The invention relates to the field of medical detection, in particular to a fecal DNA extraction and preservation solution, a preservation tube, a kit and an extraction method. A fecal DNA extraction and preservation solution comprises 0.20-0.30M EDTA, 0.2-0.8% alanine, 20-30% ethanol, 0.2-0.8% sodium dodecyl sulfate, 1.0-5.0% polyvinylpyrrolidone and has a pH value of 10.0-11.0; the above percentages are mass percentages. Under the condition of high EDTA/alkalinity, the preservation solution adopts alcohol as a bactericide, adopts alanine as a buffer system in a high-alkalinity environment, adopts sodium dodecyl sulfate and polyvinylpyrrolidone as a detergent and a detergent in a synergistic manner, can prolong the preservation time of the excrement, immediately protects the excrement after the excrement is separated from the body, and ensures the integrity of genes.

Description

Excrement DNA extraction kit

Technical Field

The invention relates to the field of medical detection, in particular to a fecal DNA extraction and preservation solution, a preservation tube, a kit and an extraction method.

Background

Colorectal cancer is the most common malignancy of the digestive tract, second to gastric and esophageal cancers. The incidence of colorectal cancer has increased over the last two decades, and the age of onset tends to be aging. Colorectal cancer is second only to lung cancer in developed western countries. Recent studies have shown that mutations in several genes such as K-ras, P53, APC, MMR, etc., lead to transformation of normal colonic mucosal epithelial cells into tumor cells (Vogelstein, B.and Kinzler, K.W., 1993), and the development of mutations in these genes has made it possible to diagnose colorectal cancer by fecal DNA detection. Whether the human genome DNA in the excrement can be efficiently extracted or not becomes a key factor influencing the diagnosis accuracy.

The fecal composition is complex, and contains a large amount of components which can generate inhibition effect on human genome DNA detection, such as polysaccharide, cholic acid and the like, and most fecal DNA is derived from intestinal bacteria, and the content of human DNA is very low. Therefore, the collected fecal samples need to be stored as soon as possible, and the currently common storage method is generally low-temperature treatment, namely freezing storage. The freezing method has complex operation and high cost, and cannot quickly and conveniently collect samples.

In addition, the kit method is the simplest and most convenient method with wide clinical application at present, and commercial fecal DNA extraction kits are various in types and different in performance, but the fecal human genome DNA extraction efficiency is different. In addition, clinical feces samples are large in quantity, DNA extraction cannot be performed in time after collection, and the feces DNA is seriously degraded by a large amount of nuclease contained in the feces, so that the storage time has a great influence on the extraction efficiency of the human genome DNA.

For example, the reagent kit for rapidly extracting the DNA of the excrement of Chinese invention patent (publication No. CN106967712A, published Japanese: 2017.07.21) has the following principle: pretreating the extracted excrement by using excrement treatment liquid, centrifugally removing excrement residues and adsorbing to remove part of impurities such as protein, cracking and releasing nucleic acid by using magnetic bead cracking liquid and proteinase K, specifically binding the released nucleic acid to magnetic beads mixed with specific probes, washing by using 1 round of

washing liquid

1 and 2 rounds of washing liquid 2, eluting the nucleic acid from the magnetic beads by using eluent, and discarding the eluted magnetic beads to obtain the excrement genome. The patent also focuses on how to extract rapidly, and how to preserve DNA efficiently, the scope is not considered.

For example, chinese patent publication No. (CN108866045A, published 2018.11.23) discloses a method for extracting DNA from human feces, which comprises collecting 2-5g of feces sample immediately after human excreting feces, placing the sample into a collection tube, wherein 10-15mL of feces are contained in the collection tube for preservation, and the components of the feces retention solution are as follows: 0.3-0.6M EDTA, 3-6M NaCl, 0.3-1.5M Tris. The Chinese invention patent (publication No. CN107980763A Kokai Japanese 2018.05.04) discloses a feces preservation reagent, which comprises a cell buffer solution, a cell fixing agent, a metal ion chelating agent and a preservative; the cell buffer solution is Tris-HCl, and the working pH range is 8-10; the cell fixing agent is at least one selected from SDS, Triton X-100 and PEG-2000; the metal ion chelating agent is EDTA and/or EDTA-Na 2; the preservative is NaCl. Both the two technologies adopt a Tris-HCl buffer system, NaCl is used as a preservative, DNA is easy to degrade in practical application, and the extraction efficiency of human genome DNA is greatly influenced.

Disclosure of Invention

The invention aims to provide a feces DNA extraction and preservation solution, which adopts alcohol as a bactericide, alanine as a buffer system in a high-alkalinity environment and sodium dodecyl sulfate and polyvinylpyrrolidone as a detergent and a detergent under the condition of high EDTA/alkalinity, can prolong the feces preservation time, and immediately protects feces after being separated from the body, thereby ensuring the integrity of genes.

In order to achieve the purpose, the excrement DNA extraction and preservation solution comprises 0.20-0.30M EDTA, 0.2-0.8% of alanine, 20-30% of ethanol, 0.2-0.8% of sodium dodecyl sulfate, 1.0-5.0% of polyvinylpyrrolidone and has a pH value of 10.0-11.0; the above percentages are mass percentages.

The preserving fluid of the invention has the functions of each component: EDTA: as inhibitors of nucleases and as stabilizers. 2. Alanine: provides a buffer system with high alkaline environment. 3. Alcohol: has strong sterilization effect and can fix the sample material. SDS, and (4): detergents, anionic surfactants, detergents. PVP and detergent. Meanwhile, each component of the invention has synergistic effect, can prolong the preservation time of the excrement, immediately protect the excrement after being separated from the body and ensure the integrity of the gene.

As a specific embodiment, the preservation solution comprises 0.25M EDTA, 0.5% alanine, 25% ethanol, 0.5% sodium lauryl sulfate, 2.0% polyvinylpyrrolidone, pH 10.5; the above percentages are mass percentages.

Another object of the present invention is to provide a fecal DNA extracting and preserving tube containing the preserving fluid.

The invention also aims to provide a fecal DNA extraction kit, which comprises a fecal storage solution, a lysis solution, a binding buffer solution, a washing solution and an eluent, wherein the fecal storage solution adopts the storage solution.

As scheme A, the lysis binding solution contains: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1.0% sodium dodecyl sulfate, pH 8.9; the binding buffer solution is isopropanol with the mass percentage concentration of 80%; the washing liquid comprises a washing liquid I and a washing liquid II, wherein the washing liquid I contains: 3M GuHCl, 10mM Tris, 50% ethanol, pH 8.0; washing solution II comprises: 10mM Tris, 80% ethanol, pH 7.5; the eluent contains: 10mM Tris, 0.1mM EDTA, pH 9.0.

As scheme B, the lysis binding solution contains: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1.0% dodecyl-N-betaine, pH 8.9; the binding buffer solution is isopropanol with the mass percentage concentration of 80%; the washing solution contains: 3M GuHCl, 10mM Tris, 50% ethanol, sodium bicarbonate 35mM, pH 9.0; the eluent contains: 10mM Tris, 0.1mM EDTA, pH 9.0. At present, 2-4 times of unequal washing are needed in DNA extraction by a magnetic bead method, time is consumed, the supernatant is required to be subjected to more precise operation after washing, pollution is easy to generate/the supernatant is not suitable for field operation outside a laboratory), and the extraction effect is also greatly improved, the washing times are found by an applicant to be difficult to reduce, mainly due to insufficient washing capacity of weak alkalinity of a washing solution on mucin and the problem of elution efficiency, the washing solution containing high-concentration sodium bicarbonate (the alkalinity is slightly stronger than that of sodium acetate and sodium chloride) is prepared, the ion balance and the alkalinity are provided, and a zwitterionic detergent dodecyl-N-betaine is matched for use, so that one-step washing can be realized (the applicant patent: application No.: 2019102093130, described in detail).

Another object of the invention is to provide a method for extracting fecal DNA by using the kit.

As a specific embodiment, the kit of the method a protocol, the method comprising the steps of:

1) adding 0.2g of excrement into a centrifugal tube containing 1ml of excrement storage solution, and shaking and uniformly mixing;

2) heating the centrifugal tube to 70 ℃, and performing vortex oscillation for 20 min;

3) centrifuging the centrifuge tube at 12000rpm for 5min, and discarding the supernatant to obtain precipitate;

4) adding 1ml of lysis solution, 10ul of Enhancer and 15ul of Inhibitor Remover into the precipitate, and performing vortex oscillation to obtain suspension;

5) heating the centrifugal tube to 55 ℃, and shaking for 20 min;

6) centrifuging the tube at 12000rpm, collecting supernatant, 10ul magnetic beads, and shaking at 65 deg.C for 5 min;

7) adding a washing solution I to wash for two times;

8) washing twice by adding a washing solution II;

9) adding eluent for elution, shaking for 5 minutes at the temperature of 60 ℃, then placing the centrifugal tube on a magnetic separation frame for 2 minutes, and transferring the supernatant into another clean centrifugal tube by using a gun head; the resulting DNA was recovered and stored at-20 ℃.

As a specific embodiment, the kit of the method B protocol comprises the following steps:

5) heating the centrifugal tube to 55 ℃, and shaking for 20 min;

7) taking out the centrifugal tube from the magnetic separation frame, adding 1000ul of washing liquid, carrying out vortex oscillation on the centrifugal tube for 5-10 times to fully mix the magnetic beads, then placing the centrifugal tube on the magnetic separation frame for 2 minutes to enable the magnetic beads in the centrifugal tube to be completely adsorbed, and carefully using a liquid-transferring gun to transfer all supernatant without touching precipitation;

8) adding eluent for elution, shaking for 5 minutes at the temperature of 60 ℃, then placing the centrifugal tube on a magnetic separation frame for 2 minutes, and transferring the supernatant into another clean centrifugal tube by using a gun head; the resulting DNA was recovered and stored at-20 ℃.

Another purpose of the invention is to provide the application of the preservation solution or the preservation tube or the excrement DNA extraction kit in gene screening and forensic identification.

Due to the adoption of the technical scheme, the invention has the following characteristics:

(1) the high EDTA/alkaline preservation solution can prolong the preservation time of the excrement, and the excrement is immediately protected after being separated from the body, so that the integrity of genes is ensured;

(2) the lysis effect of the lysis solution is rapid and stable, the sample nucleic acid can be released at normal temperature, and the released DNA is combined, so that two functions of lysis and combination are completed in one step; time and labor are saved, the efficiency is improved, and the operation on the machine is convenient;

(3) the method is suitable for manual extraction, can be matched with a nucleic acid extractor in a micropore plate in an automatic mode for use, has high extraction purity, is simple, convenient and safe to operate, and effectively avoids the problems of easy degradation and poor extraction quality in the excrement extraction process;

(4) high concentration, purity: the DNA is combined with magnetic beads, the concentration and purity of the extracted DNA are high, and the DNA can be directly used for downstream experiments;

(5) the kit has good stability;

(6) the reagent does not contain toxic solvents such as phenol, chloroform and the like, and the operation is safe and environment-friendly.

Drawings

FIG. 1 is an electrophoresis picture of DNA extracted from a stool sample of five persons according to the method of example 1.

FIG. 2 is a graph of DNA concentrations extracted from five stool samples according to the method of example 1.

FIG. 3 is an electrophoresis picture of DNA samples of stools placed at room temperature for 1d, 3d, 7d, and 14d in example 1.

FIG. 4 is a graph showing the concentration of DNA extracted at different storage times in example 1.

FIG. 5 is an electrophoresis picture of DNA extracted from a stool sample of five persons in the method of example 2.

FIG. 6 is an electrophoresis image of DNA samples of feces set at room temperature for 1d, 3d, 7d, and 14d in example 2.

FIG. 7 is a photograph showing the electrophoresis of a part of the amplification product obtained by the method of example 1.

Detailed Description

Primary reagents and instruments

Tris-HCl, Tris, EDTA, Alanine, NaCl, SDS, PVP and GuHCl are provided by sigma;

ethanol and isopropanol are produced by the national medicine group;

the magnetic beads used in the detection method of the present application are produced by Qiagen (the magnetic beads taken from the Magattract DNA Kit, the enriching Kit are self-contained).

Detecting the origin of a sample

The fecal sample used in the verification of the DNA extraction effect of the kit of the application is from a sample normally collected by a cooperative medical detection institution.

Example 1

Firstly, the formula is as follows:

1. feces preservative solution containing 0.25M EDTA, 0.5% alanine, 25% ethanol, 0.5% sodium dodecyl sulfate, and 2% PVP; pH10.5;

2. lysis solution of 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1% sodium dodecyl sulfate and 20% isopropanol; pH 8.9;

3. wash I3M GuHCl, 10mM Tris, 50% ethanol; pH 8.0;

4. wash II 10mM Tris, 80% ethanol; pH 7.5;

5、elution:10mM Tris，0.1mM EDTA；pH 9.0。

secondly, a method for taking excrement DNA, which comprises the following steps:

5) heating the centrifugal tube to 55 ℃, and shaking for 20 min;

7) adding a washing solution I to wash for two times;

8) washing twice by adding a washing solution II;

Example 2

Firstly, the formula is as follows:

3. wash 3M GuHCl, 10mM Tris, 50% ethanol, sodium bicarbonate 35mM, pH 9.0;

4、elution:10mM Tris，0.1mM EDTA；pH 9.0。

5) heating the centrifugal tube to 55 ℃, and shaking for 20 min;

Test examples

1. Determination of DNA solution concentration and purity

Adding DNA2ul to be detected into a cuvette, adding double distilled water to 100ul, fully and uniformly mixing, and detecting OD260, OD280 and the ratio of OD260 to OD280 of the DNA by a UV-7504 ultraviolet visible light photometer.

DNA concentration calculation formula: DNA concentration OD260 × dilution × 50/1000(ug/u 1);

DNA purity: the ratio of OD260/OD280 is 1.8-2.0, and the DNA purity is considered to be good; the ratio of OD260/OD280 is obviously more than 2.0, and RNA pollution or DNA fragment breakage is considered to be possible in DNA; the OD260/OD280 ratio was clearly below 1.8, suggesting that there may be protein in the DNA or phenol in the residual extraction.

2. 2% agarose gel electrophoresis to determine the concentration and integrity of the extracted DNA

5ul of the extracted DNA + lul Loading buffer (total volume 6u1) was added to 2% agarose gel, electrophoresed for 30min, the purity of the band was observed and the concentration was estimated in a KH-UVI type ultraviolet transmission analyzer, and a photograph was taken and recorded.

3. Confirmation of human genomic DNA

Since human genome DNA in feces only accounts for about 1% of the total amount of DNA, the experimental procedure provided in example 1 only increases the proportion of human genome in the extracted DNA as much as possible, but does not completely exclude non-human DNA. The amplification of human specific genes was generally used for judgment, and the beta-actin gene was used in this experiment according to literature reports.

1) Amplification of beta-actin gene:

the preparation of the human genome beta-actin gene amplification reaction solution and the setting of the reaction conditions are shown in the following tables 1 and 2, and the primers, annealing temperature and products are shown in the following table 3.

TABLE 1 beta-actin gene amplification reaction solution composition

TABLE 2 amplification reaction conditions for beta-actin gene

TABLE 3 primers, annealing temperature and product size for PCR reactions

2) And (3) detecting a PCR amplification result:

the amplified product was electrophoresed through 2% agarose gel, and the purity and concentration of the amplified band were evaluated in KH-UVI type ultraviolet transmission analyzer, and a photograph was taken and recorded.

4. Results

1) Extraction results of fecal DNA

Fecal DNA was extracted as in example 1, and the resulting product was converted to dsDNA concentration using a spectrophotometer to measure OD at 260rim wavelength. 100 stool samples were extracted, and the amount of DNA extracted from 200mg stools varied from 20-105ug, averaging 56 ug. The ratio of OD260/OD280 of DNA is less than 18 for 8 cases, M of 1.8-2.0 for 90 cases, and more than 2.0 for 2 cases.

Five groups are taken, 2% agarose gel electrophoresis is carried out on the extracted fecal DNA, the electrophoresis picture is shown in figure 1, and the DNA concentration is shown in figure 2. A group of samples of stools placed at normal temperature for 1d, 3d, 7d and 14d adopt 2% agarose gel electrophoresis, the electrophoresis picture is shown in figure 3, and the DNA concentration is shown in figure 4.

Fecal DNA was extracted as in example 2, and the resulting product was converted to dsDNA concentration using a spectrophotometer to measure OD at 260rim wavelength. 100 stool samples were extracted, and the amount of DNA extracted from 200mg stools varied from 30-85ug, averaging 45 ug. The ratio of OD260/OD280 of DNA was 11 cases less than 18, 86 cases of M between 1.8 and 20, and 3 cases of M greater than 2.0.

Five groups are taken, 2% agarose gel electrophoresis is carried out on the extracted excrement DNA, and the electrophoresis picture is shown in figure 5. A group of samples of stools placed at normal temperature for 1d, 3d, 7d and 14d adopt 2% agarose gel electrophoresis, and an electrophoresis picture is shown in figure 6.

2) Confirmation of human genomic DNA

In order to confirm the existence of human genome, the fecal DNA extracted from the sample in example 1 is subjected to PCR reaction to amplify human gene beta-actin fragment, the amplified product is subjected to 15% agarose gel electrophoresis, and the target fragment is amplified and then subjected to sulfite treatment and MS-PCR reaction. The human gene beta-actin can be amplified from all 100 cases of excrement DNA, and the electrophoresis result of partial amplification products is shown in figure 7.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A feces DNA extraction and preservation solution is characterized by comprising 0.20-0.30M of EDTA, 0.2-0.8% of alanine, 20-30% of ethanol, 0.2-0.8% of sodium dodecyl sulfate, 1.0-5.0% of polyvinylpyrrolidone and having a pH value of 10.0-11.0; the above percentages are mass percentages.

2. The preservation solution according to claim 1, characterized in that it comprises 0.25M EDTA, 0.5% alanine, 25% ethanol, 0.5% sodium lauryl sulfate, 2.0% polyvinylpyrrolidone, pH 10.5; the above percentages are mass percentages.

3. A fecal DNA extraction storage tube containing the storage solution of claim 1 or 2.

4. A fecal DNA extraction kit, characterized in that the kit comprises a fecal storage solution, a lysis solution, a binding buffer solution, a washing solution and an eluent, wherein the fecal storage solution is the storage solution of claim 1 or 2.

5. The kit of claim 4, wherein the lysis buffer and binding buffer comprise: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1.0% sodium lauryl sulfate, 20% isopropanol, pH 8.9; the washing liquid comprises a washing liquid I and a washing liquid II, wherein the washing liquid I contains: 3M GuHCl, 10mM Tris, 50% ethanol, pH 8.0; washing solution II comprises: 10mM Tris, 80% ethanol, pH 7.5; the eluent contains: 10mM Tris, 0.1mM EDTA, pH 9.0.

6. The kit of claim 4, wherein the lysis conjugate comprises: 0.1M Tris-HCl, 15mM EDTA, 1M NaCl, 3M GuSCN, 1.0% dodecyl-N-betaine, 20% isopropanol, pH 8.9; the washing solution contains: 3M GuHCl, 10mM Tris, 50% ethanol, sodium bicarbonate 35mM, pH 9.0; the eluent contains: 10mM Tris, 0.1mM EDTA, pH 9.0.

7. A method for extracting fecal DNA according to the kit of claim 4 or 5 or 6.

8. The method of claim 7, wherein the method uses the kit of claim 5, comprising the steps of:

5) heating the centrifugal tube to 55 ℃, and shaking for 20 min;

7) adding a washing solution I to wash for two times;

8) washing twice by adding a washing solution II;

9. The method of claim 7, wherein the method uses the kit of claim 6, comprising the steps of:

5) heating the centrifugal tube to 55 ℃, and shaking for 20 min;