阅读说明：本技术 一种尿液基因组提取试剂盒及使用方法 (Urine genome extraction kit and use method ) 是由 王文婧 张炳为 于 2021-10-15 设计创作，主要内容包括：本申请提供的尿液基因组提取试剂盒,包括：裂解液、第一洗涤液、第二洗涤液和洗脱液；其中,所述裂解液包括盐酸胍、尿素、乙酸铵及曲拉通X-100,所述裂解结合液中盐酸胍的浓度为1～1.5mol/L,尿素的浓度为50～100mmol/L,乙酸铵的浓度为1～50mmol/L、曲拉通X-100的浓度为10％～20％(v/v),上述试剂盒操作简便,无需对尿液样本进行预处理,整个过程室温操作时间短,提取效率高。(The application provides a urine genome extraction kit, includes: lysis solution, first washing solution, second washing solution and eluent; the lysis solution comprises guanidine hydrochloride, urea, ammonium acetate and Triton X-100, the concentration of the guanidine hydrochloride in the lysis binding solution is 1-1.5mol/L, the concentration of the urea is 50-100 mmol/L, the concentration of the ammonium acetate is 1-50 mmol/L, and the concentration of the Triton X-100 is 10-20% (v/v), the kit is simple and convenient to operate, a urine sample does not need to be pretreated, the room temperature operation time in the whole process is short, and the extraction efficiency is high.)

1. A urine genome extraction kit, comprising: lysis solution, first washing solution, second washing solution and eluent; the cracking solution comprises guanidine hydrochloride, urea, ammonium acetate and triton X-100, the concentration of the guanidine hydrochloride in the cracking binding solution is 1-1.5mol/L, the concentration of the urea is 50-100 mmol/L, the concentration of the ammonium acetate is 1-50 mmol/L, and the concentration of the triton X-100 is 10-20% (v/v).

2. The urine genome extraction kit according to claim 1, wherein the first washing solution comprises the following raw materials: guanidine hydrochloride, tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, hydrochloric acid and isopropanol.

3. The urine genome extraction kit according to claim 2, wherein the concentration of guanidine hydrochloride in the first washing solution is 3 to 6mol/L, the concentration of tris (hydroxymethyl) aminomethane is 15 to 25mmol/L, the concentration of ethylenediaminetetraacetic acid is 2 to 3mmol/L, the volume concentration of hydrochloric acid is 0.1 to 0.2%, and the volume concentration of isopropanol is 20 to 30%.

4. The urine genome extraction kit according to claim 1, wherein the second washing solution comprises the following raw materials: sodium acetate, ammonium acetate and glacial acetic acid.

5. The urine genome extraction kit according to claim 4, wherein the concentration of sodium acetate in the second washing solution is 150 to 200mmol/L, the concentration of ammonium acetate is 200 to 300mmol/L, the volume concentration of glacial acetic acid is 1 to 2%, and the volume concentration of ethanol is 5 to 10%.

6. The urine genome extraction kit according to claim 1, wherein the eluent comprises tris and edta.

7. The urine genome extraction kit according to claim 1, wherein the concentration of tris (hydroxymethyl) aminomethane in the eluent is 5 to 15mmol/L, and the concentration of ethylenediaminetetraacetic acid is 1 to 2 mmol/L.

8. The use method of the urine genome extraction kit according to claim 1, comprising the following steps:

centrifuging the urine sample to collect precipitate, adding the lysate and the proteinase K solution into the precipitate, placing the precipitate in a centrifugal tube, and uniformly mixing to obtain a first mixed solution;

placing the first mixed solution in a metal bath for incubation, then carrying out centrifugal treatment, and collecting supernatant;

adding isopropanol into the supernatant, uniformly mixing, and standing to obtain a second mixed solution;

transferring the second mixed solution to a DNA preparation tube for centrifugation, and discarding the filtrate;

placing the DNA preparation tube back into the centrifuge tube, adding the first washing solution, centrifuging and discarding the filtrate;

placing the DNA preparation tube back into the centrifuge tube, adding the second washing solution, centrifuging and discarding the filtrate;

and (3) drying the DNA preparation tube, adding the eluent to elute nucleic acid, centrifuging, collecting supernate and storing.

9. The application method of the urine genome extraction kit according to claim 8, wherein in the steps of air-drying the DNA preparation tube, adding the eluent to elute nucleic acid, centrifugally collecting and storing the supernatant, the supernatant is stored at-18 to-22 ℃.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a urine genome extraction kit and a use method thereof.

Background

Bladder cancer (BCa) is the most common malignancy of the urinary system, with approximately 549,393 new cases and 199,922 deaths worldwide each year. While about 75% of patients suffer from non-muscle invasive bladder cancer (NMIBC), with 70% of the tumors recurring, and therefore, patients suffering from NMIBC require frequent treatment and monitoring, which results in higher treatment costs. The current gold standard for monitoring BCa recurrence is cystoscopy and cytology, but is invasive and costly, whereas urine cytology is highly specific but lacks sensitivity. Fluorescence In Situ Hybridization (FISH) is highly sensitive and widely used in conventional BCa clinical assays, but is less sensitive to low grade or small tumors. DNA methylation is an important epigenetic regulator of gene expression, and abnormal expression often results in defective gene expression, and in many tumors, increased methylation of cancer suppressor genes is one of the early events. Therefore, the bladder cancer can be screened and diagnosed, the recurrence and metastasis can be judged, the selection of a treatment scheme can be carried out, and the like by detecting the abnormal methylation of the gene.

The DNA in urine comes from the cast-off cells in the urethra, and the basic research and clinical diagnosis of molecular biology by using urine DNA molecules have many special advantages: 1) the urine collection is non-invasive and non-invasive. 2) Extraction of DNA from urine is much simpler than extraction of DNA from blood.

Disclosure of Invention

In view of the above, there is a need to provide a urine genome extraction kit and a method for using the same, which are specially used for extracting genomic DNA from urine, aiming at the defects existing in the prior art.

In order to solve the problems, the invention adopts the following technical scheme:

the application provides a urine genome extraction kit, includes: lysis solution, first washing solution, second washing solution and eluent; the cracking solution comprises guanidine hydrochloride, urea, ammonium acetate and triton X-100, the concentration of the guanidine hydrochloride in the cracking binding solution is 1-1.5mol/L, the concentration of the urea is 50-100 mmol/L, the concentration of the ammonium acetate is 1-50 mmol/L, and the concentration of the triton X-100 is 10-20% (v/v).

In some of these embodiments, the first wash solution comprises the following raw materials: guanidine hydrochloride, tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, hydrochloric acid and isopropanol.

In some embodiments, the concentration of guanidine hydrochloride in the first washing solution is 3-6 mol/L, the concentration of tris (hydroxymethyl) aminomethane is 15-25 mmol/L, the concentration of ethylene diamine tetraacetic acid is 2-3 mmol/L, the volume concentration of hydrochloric acid is 0.1-0.2%, and the volume concentration of isopropanol is 20-30%.

In some embodiments, the second wash solution comprises the following materials: sodium acetate, ammonium acetate and glacial acetic acid.

In some embodiments, the concentration of sodium acetate in the second washing solution is 150-200 mmol/L, the concentration of ammonium acetate is 200-300 mmol/L, the volume concentration of glacial acetic acid is 1% -2%, and the volume concentration of ethanol is 5-10%.

In some of these embodiments, the eluent comprises tris and edta.

In some embodiments, the concentration of tris (hydroxymethyl) aminomethane in the eluent is 5-15 mmol/L, and the concentration of ethylenediaminetetraacetic acid is 1-2 mmol/L.

In addition, the application also provides a using method of the urine genome extraction kit, which comprises the following steps:

centrifuging the urine sample to collect precipitate, adding the lysate and the proteinase K solution into the precipitate, placing the precipitate in a centrifugal tube, and uniformly mixing to obtain a first mixed solution;

placing the first mixed solution in a metal bath for incubation, then carrying out centrifugal treatment, and collecting supernatant;

adding isopropanol into the supernatant, uniformly mixing, and standing to obtain a second mixed solution;

transferring the second mixed solution to a DNA preparation tube for centrifugation, and discarding the filtrate;

placing the DNA preparation tube back into the centrifuge tube, adding the first washing solution, centrifuging and discarding the filtrate;

placing the DNA preparation tube back into the centrifuge tube, adding the second washing solution, centrifuging and discarding the filtrate;

and (3) drying the DNA preparation tube, adding the eluent to elute nucleic acid, centrifuging, collecting supernate and storing.

In some embodiments, the step of adding the eluent to elute nucleic acids after the DNA preparation tube is air-dried, and collecting and storing a supernatant by centrifugation, the supernatant is stored at-18 to-22 ℃.

The technical scheme adopted by the application has the following effects:

the application provides a urine genome extraction kit, includes: lysis solution, first washing solution, second washing solution and eluent; the lysis solution comprises guanidine hydrochloride, urea, ammonium acetate and Triton X-100, the concentration of the guanidine hydrochloride in the lysis binding solution is 1-1.5mol/L, the concentration of the urea is 50-100 mmol/L, the concentration of the ammonium acetate is 1-50 mmol/L, and the concentration of the Triton X-100 is 10-20% (v/v), the kit is simple and convenient to operate, a urine sample does not need to be pretreated, the room temperature operation time in the whole process is short, and the extraction efficiency is high.

In addition, the urine genome extraction kit provided by the application can extract a large volume of urine samples, 10-50ml of urine samples can be extracted by using the kit, and the extracted DNA is pure and can be directly used for PCR, DNA methylation identification, early cancer detection and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention or in the description of the prior art will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a flowchart of steps of a method for using a urine genome extraction kit provided in an embodiment of the present application.

FIG. 2 is a graph showing the amplification of beta-actin gene in the samples mentioned in examples 1 and 2 of the present application.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "horizontal", "inside", "outside", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.

The application provides a urine genome extraction kit, includes: lysis solution, first washing solution, second washing solution and eluent; the cracking solution comprises guanidine hydrochloride, urea, ammonium acetate and triton X-100, the concentration of the guanidine hydrochloride in the cracking binding solution is 1-1.5mol/L, the concentration of the urea is 50-100 mmol/L, the concentration of the ammonium acetate is 1-50 mmol/L, and the concentration of the triton X-100 is 10-20% (v/v).

In some of these embodiments, the first wash solution comprises the following raw materials: guanidine hydrochloride, tris (hydroxymethyl) aminomethane, ethylenediamine tetraacetic acid, hydrochloric acid and isopropanol.

Furthermore, the concentration of guanidine hydrochloride in the first washing liquid is 3-6 mol/L, the concentration of tris (hydroxymethyl) aminomethane is 15-25 mmol/L, the concentration of ethylene diamine tetraacetic acid is 2-3 mmol/L, the volume concentration of hydrochloric acid is 0.1-0.2%, and the volume concentration of isopropanol is 20-30%.

In some embodiments, the second wash solution comprises the following materials: sodium acetate, ammonium acetate and glacial acetic acid.

Further, the concentration of sodium acetate in the second washing liquid is 150-200 mmol/L, the concentration of ammonium acetate is 200-300 mmol/L, the volume concentration of glacial acetic acid is 1% -2%, and the volume concentration of ethanol is 5-10%.

It will be appreciated that the inclusion of both ammonium acetate and sodium acetate in the second wash solution facilitates the dissolution of residual protein.

In some of these embodiments, the eluent comprises tris and edta.

Furthermore, the concentration of the trihydroxymethyl aminomethane in the eluent is 5-15 mmol/L, and the concentration of the ethylene diamine tetraacetic acid is 1-2 mmol/L.

It can be understood that the elution solution can be more stably dissolved by adding EDTA to remove the influence of interfering ions.

Referring to fig. 1, the present application further provides a method for using the urine genome extraction kit, comprising the following steps:

step S110: and centrifuging the urine sample to collect precipitate, adding the lysate and the proteinase K solution into the precipitate, placing the precipitate in a centrifuge tube, and uniformly mixing to obtain a first mixed solution.

Specifically, a fresh 10-50ml urine sample is provided, centrifuged at 3000 Xg, 1ml of the precipitate is left and transferred to a 1.5ml centrifuge tube, centrifuged at 12000 Xg for 1min, the supernatant is discarded, the precipitate is collected, and 500. mu.L of lysate and 20. mu.L of proteinase K are added to the precipitate.

Step S120: and placing the first mixed solution in a metal bath for incubation, then carrying out centrifugal treatment, and collecting supernatant.

Specifically, the mixture was lysed in a 85 ℃ metal bath for 15min, and the lysed solution was centrifuged at 12000 Xg to collect the supernatant and transferred to a clean 1.5ml centrifuge tube.

Step S130: adding isopropanol into the supernatant, uniformly mixing, and standing to obtain a second mixed solution.

Specifically, 0.5-fold volume of isopropyl alcohol was added to the collected supernatant and mixed well to obtain a second mixed solution.

Step S140: the second mixture was transferred to a DNA preparation tube and centrifuged, and the filtrate was discarded.

Specifically, the second mixture was transferred to a prepared DNA preparation tube and centrifuged at 6000 Xg for 1min and the filtrate was discarded.

Step S150: the DNA preparation tube was placed back into the centrifuge tube and the first wash was added and centrifuged and the filtrate discarded.

Specifically, the DNA preparation tube was returned to the centrifuge tube, 500. mu.L of the first wash solution was added, and the mixture was centrifuged at 12000 Xg for 1 min.

Step S160: place the DNA preparation tube back into the centrifuge tube and then add the second wash solution and centrifuge and discard the filtrate.

Specifically, the DNA preparation tube is placed back into the centrifuge tube, 700. mu.L of a second washing solution (12000 Xg) is added, centrifugation is carried out for 1min, and the filtrate is discarded and repeated once.

Step S170: and (3) drying the DNA preparation tube, adding the eluent to elute nucleic acid, centrifuging, collecting supernate and storing.

Specifically, the air-dried DNA preparation tube is placed in another clean 1.5ml centrifuge tube, 50 μ L of eluent is added to the center of the preparation tube membrane, the mixture is kept stand at room temperature for 2min, and then is centrifuged at 12000 Xg for 2min to elute DNA, and the eluent is collected and stored.

In some embodiments, the step of adding the eluent to elute nucleic acids after the DNA preparation tube is air-dried, and collecting and storing a supernatant by centrifugation, the supernatant is stored at-18 to-22 ℃.

Above-mentioned urine genome extraction kit need not to carry out preliminary treatment to the urine sample in the use, reduces the extraction time, does not use toxic reagent in the extraction process, and the operation is safe, can high-efficiently extract genome DNA, and the extraction result can be used to the further experiment of low reaches.

In addition, the urine genome extraction kit provided by the application can extract a large volume of urine samples, 10-50ml of urine samples can be extracted by using the kit, and the extracted DNA is pure and can be directly used for PCR, DNA methylation identification, early cancer detection and the like.

The technical solution of the present application is described in detail below with reference to specific embodiments.

Example 1

The embodiment 1 of the application provides a kit for extracting cell genome DNA in urine, which comprises a lysis binding solution, a first washing solution, a second washing solution and an eluent.

Wherein, the lysis solution comprises the following raw materials: guanidine hydrochloride, urea, sodium acetate and triton X-100; the concentration of guanidine hydrochloride is 1.25mol/L, the concentration of urea is 75mmol/L, the concentration of sodium acetate is 40mmol/L, and the concentration of triton X-100 is 18.75% (v/v).

The concentration of guanidine hydrochloride in the first washing liquid is 4mol/L, the concentration of tris (hydroxymethyl) aminomethane is 20mmol/L, the concentration of ethylene diamine tetraacetic acid is 2.5mmol/L, the volume concentration of hydrochloric acid is 0.15%, and the volume concentration of isopropanol is 25%;

the concentration of sodium acetate in the second washing solution is 180mmol/L, the concentration of ammonium acetate is 250mmol/L, and the volume concentration of glacial acetic acid is 1.5%;

the concentration of the trihydroxymethyl aminomethane in the eluent is 10mmol/L, and the concentration of the ethylene diamine tetraacetic acid is 1.5 mmol/L; the pH of the eluent was 8.

3 human urine samples were extracted using the kit of example 1, following the following procedure:

1) preparation before experiment:

(1) when the reagent bottle is used for the first time, absolute ethyl alcohol with a specified volume is added into the reagent bottle;

(2) preparing a temperature bath at 85 ℃;

(3) checking whether the cracking binding solution has precipitate before use, and if the precipitate appears, heating the cracking binding solution in a warm bath at 70 ℃ until the precipitate is completely dissolved for use;

(4) a20 mg/ml proteinase K solution was prepared.

2) The experimental steps are as follows:

(1) preparing 3 centrifuge tubes of 50ml, and collecting three urine samples of 30ml respectively; centrifuging three samples at 3000 Xg for 5min, reserving 1ml of precipitate, transferring the precipitate into a 1.5ml centrifuge tube, centrifuging at 12000 Xg, discarding supernatant, and collecting precipitate;

(2) adding 500 mu L of lysis solution and 20 mu L of proteinase K solution into the precipitate, and uniformly mixing;

(3) the mixed solution in the step (2) is put into a metal bath at 85 ℃ for cracking for 15 min;

(4) the lysed solution was centrifuged at 12000 Xg to collect the supernatant and transferred to a clean 1.5ml centrifuge tube;

(5) adding 0.5 volume times of isopropanol into the collected supernatant and uniformly mixing;

(6) transferring the mixed solution obtained in the step (5) to a prepared DNA preparation tube, centrifuging at 6000 Xg for 1min, and discarding the filtrate;

(7) placing the prepared tube back into the original centrifuge tube, adding 500 μ L of the first washing solution, and centrifuging at 12000 Xg for 1 min;

(8) placing the preparation tube back into the original centrifuge tube, adding 700 μ L of second washing solution, centrifuging at 12000 Xg for 1min, discarding the filtrate, and repeating the steps;

(9) placing the air-dried DNA preparation tube in another clean 1.5ml centrifuge tube, adding 50 μ L of eluent in the center of the preparation tube membrane, standing at room temperature for 2min, centrifuging at 12000 Xg for 2min to elute DNA, collecting eluent and storing.

And (5) after the extraction is finished, carrying out quantitative and purity detection on the DNA. mu.L of each sample was sampled, and the values of A260 and A280 of the samples were measured using Quawell ultramicrospectrophotometer Q3000 using the above-mentioned eluent as a blank, and the purity of the nucleic acid was evaluated by the ratio.

Example 2

Preparing 3 centrifuge tubes of 50ml, and collecting three urine samples of 30ml respectively; three samples were centrifuged at 3000 Xg for 5min, leaving 1ml of pellet and transferred to 1.5ml centrifuge tubes where the pellet was collected by centrifugation at 12000 Xg and discarding the supernatant. 3 urine samples were extracted using a common commercially available urine genome extraction kit.

And (5) after the extraction is finished, carrying out quantitative and purity detection on the DNA. mu.L of each sample was sampled, and the values of A260 and A280 of the samples were measured using Quawell ultramicrospectrophotometer Q3000 using the above-mentioned eluent as a blank, and the purity of the nucleic acid was evaluated by the ratio.

For the urine DNA extracted in the above examples 1 and 2, the beta-actin gene was detected by real-time fluorescent quantitative PCR, and the Ct value results are shown in the following table.

TABLE 1 concentration of DNA extracted from different urine samples and Ct value detected by PCR with the kit of the present invention

As can be seen from Table 1, the cell genome DNA in urine can be well extracted by using the kit of the present invention in example 1, the concentration and purity of the extracted DNA are high, C (ng/. mu.L) in the table indicates the concentration of the extracted DNA, the A260/280 ratio indicates the degree of contamination of organic matters such as protein, and the A260/280 ratio of the high quality sample should be between 1.8 and 2.0. Samples 1-3 were extracted by the above method, and it can be seen that 260/280 ratios of 3 samples are all greater than 1.8, indicating that there is no contamination by substances such as protein in the samples, and the concentration of DNA extracted by using 30mL urine is all greater than 80 ng/. mu.L. The extraction results were superior to those of example 2, i.e., a common commercial kit, in both the extraction concentration and the extraction purity. From the result of fluorescent quantitative PCR, the Ct value of the beta-actin gene amplification of the sample extracted in the example 1 is 2-3 lower than the Ct value of the sample extracted in the example 2, the larger the Ct value is, the lower the content of the target DNA is, the genomic DNA in urine can be effectively extracted by the kit, and the extraction amount can be used in the next experiment.

Examples 3 to 6

Examples 3-6 differ from example 1 in the concentration of guanidine hydrochloride in the lysate, as shown in Table 2

TABLE 2 guanidine hydrochloride concentration in lysates of examples 3-7

	Example 3	Example 4	Example 5	Example 6	Example 7
						Concentration of guanidine hydrochloride	0.5mol/L	1mol/L	1.5mol/L	2mol/L	2.5mol/L

Collecting 30ml urine samples respectively; the sample was centrifuged at 3000 Xg for 5min, leaving 1ml of pellet and transferred to a 1.5ml centrifuge tube where the pellet was collected by centrifugation at 12000 Xg and discarding the supernatant. Samples were extracted using the kit. The procedure of example 1 was repeated except that the concentration of guanidine hydrochloride in the lysate was different, and the DNA was quantitatively determined and the purity thereof was measured after the extraction. mu.L of each sample was sampled, and the values of A260 and A280 of the samples were measured using Quawell ultramicrospectrophotometer Q3000 using the above-mentioned eluent as a blank, and the purity of the nucleic acid was evaluated by the ratio. The results are shown in Table 3.

Table 3 examples 3-7 sample extraction results

As is clear from Table 3, the results of the urine samples extracted in examples 3 to 7 showed that the concentration of the extracted sample was 60 to 90 ng/. mu.L, the nucleic acid A260/280 was 1.80 or more, and the samples were free from contamination with substances such as proteins. The concentrations of the nucleic acids extracted in examples 4 and 5 were higher than those extracted in examples 3, 6 and 7, and there was no significant difference between the purities of the samples extracted in examples 3 to 7. The results show that the optimal concentration of guanidine hydrochloride in the lysis solution of the kit is 1-1.5mol/L, the sample lysis effect is good, and the extracted nucleic acid can be used in the next experiment.

The above are merely examples of the present application and are not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.