CN113621607A

CN113621607A - Lysis solution and application thereof

Info

Publication number: CN113621607A
Application number: CN202110907790.1A
Authority: CN
Inventors: 刘一丁; 赵久茗; 王江
Original assignee: Chengdu Northen Medical Laboratory Co ltd
Current assignee: Chengdu Northen Medical Laboratory Co ltd
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-11-09

Abstract

The invention relates to the technical field of molecular biology, in particular to a lysate and application thereof. The invention has simple preparation and easy preparation of the lysis solution under the condition of ensuring good stability of the lysis solution. The lysate included water, Tris-HCl, Tween, Triton X-100, and EDTA. The lysate can ensure that the nucleic acid can be extracted without adding proteinase K in the lysis process, shorten the period of extracting sample nucleic acid by the lysate, and reduce the cost of the lysate. The method for extracting nucleic acid is rapid and simple, low in cost and sensitive in detection, and the detection result of the genome extracted by the method is similar to that of the genome extracted by a commercial kit. The reagent is used for extracting the genomic DNA of the buccal swab, the nucleic acid extraction is completed within 20 minutes, and the fluorescent quantitative PCR detection is carried out by diluting a sample by a certain multiple.

Description

Lysis solution and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a lysate and application thereof.

Background

In recent years, with the continuous maturation of the fluorescent quantitative PCR technology, the fluorescent quantitative PCR technology has been widely applied in the fields of scientific research and inspection, and becomes an essential important technology in the field of molecular biology. Especially in the process of genetic background analysis, the buccal swab is used as an important biological analysis sample, and the fluorescent quantitative PCR detection of buccal cells is frequently required.

Compared with the ordinary PCR, the fluorescent quantitative PCR detection has higher requirements on samples, but because a plurality of PCR reaction inhibitors (for example, the oral swab contains protein, fat, anticoagulant and the like), the direct fluorescent quantitative PCR reaction by using the oral swab is particularly difficult. Therefore, in clinical practice, DNA is purified from a buccal swab sample before the DNA amplification of the sample is performed.

The methods for extracting the genome from the buccal swab are very various, and examples thereof include a silica gel-mold centrifugal column adsorption method, a magnetic bead method and the like. Generally, the method for extracting nucleic acid from the oral swab comprises the following 4 processes: 1. the oral exfoliative cells are lysed by a lysis solution to release the nucleic acid material. 2. And (3) adsorbing the nucleic acid substances through a centrifugal column or nano magnetic beads specifically, and removing other impurities. 3. And washing away the chemical reagent residue and protein residue hung on the surface of the centrifugal column or the nanometer magnetic bead by using a rinsing solution. 4. Adding eluent to elute nucleic acid from the centrifugal column or nanometer magnetic bead. The extraction method has long time consumption, high cost and various steps in the nucleic acid extraction and purification process, and increases the risk of gene pollution. Moreover, the most critical of these steps is the cell lysis step, in which the extent of cell lysis determines the effect of late-stage amplification. Currently, the commonly used lysis solution mostly contains a proteinase K component, and the cell lysis by utilizing the proteinase K requires incubation at 37 ℃, so that the lysis step is added. And the need for low-temperature refrigeration and transportation in order to maintain proteinase K activity causes inconvenience in storage and transportation. On the other hand, if proteinase K is not completely removed during the purification process, the amplification reaction at the later stage is affected.

Therefore, the field is eager to develop a cracking agent without proteinase K, so that the buccal swab sample can be used as a template to perform a fluorescence quantitative PCR amplification detection method after being simply processed, and the requirement of rapid detection in scientific research and medical fields is met.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a proteinase K-free lysis solution suitable for oral cells and use thereof.

Experiments show that the lysis solution can achieve a good lysis effect on oral cells even if protease K is not added.

The lysis solution provided by the invention comprises water, Tris-HCl, Tween, Triton X-100 and EDTA.

In the lysis solution, the pH value of Tris-HCl is 8.3. In the lysate, Tween and Triton X-100 are used as detergents, so that a good lysis effect is achieved. It is verified that the cracking effect of the selected Tween-20 is due to the surfactants of other Tween series. Wherein the mass ratio of the Tween-20 to the Triton X-100 is preferably 8:1, and when the Tween-20 and the Triton X-100 are in the ratio, the amplification effect of the products obtained by cracking is optimal.

In the embodiment of the invention, the lysis solution consists of water and the following components: 10-30 mM Tris-HCl, 0.5-1 wt% Tween, 0.01-1 wt% Triton X-100 and 1-20 mM EDTA.

In some embodiments, the lysis solution consists of water and the following components: 20mM Tris-HCl, 0.8 wt% Tween-20, 0.1 wt% Triton X-100 and 10mM EDTA.

The lysate is applied to the lysis of animal cell samples.

The lysate is applied to the preparation of a kit, and the kit is an animal cell sample nucleic acid extraction kit or a fluorescence PCR pretreatment kit.

The animal cell sample is an animal oral swab sample. The animal includes a human.

The kit for extracting the nucleic acid of the animal cell sample comprises the lysate and the BSA solution. The nucleic acid is genomic DNA of an animal.

In some embodiments, the volume ratio of the lysis solution to the BSA solution is 200: 4.

In the BSA solution, the concentration of BSA is 20 mg/mL.

The invention also provides a nucleic acid extraction method of the animal cell sample, which comprises the steps of mixing a sample to be extracted with the lysate, mixing with a bovine serum albumin solution after vortex oscillation, incubating for 8-12 min at 60-70 ℃ after vortex oscillation again, incubating for 4-6 min at 90-100 ℃, and centrifuging to obtain a supernatant containing nucleic acid.

In the present invention, the sample to be extracted is a buccal swab, and the nucleic acid is genomic DNA.

In this extraction method, 200. mu.L of the lysate is mixed with each buccal swab.

The concentration of bovine serum albumin in the bovine serum albumin solution is 20mg/mL, and the volume ratio of the bovine serum albumin solution to the nucleic acid lysate is 4: 200.

performing instantaneous centrifugation before incubation at the temperature of 60-70 ℃; and (3) performing instantaneous centrifugation before incubation at 90-100 ℃. The centrifugation was carried out at 12000 rpm for 5 minutes.

In some embodiments, the incubation comprises incubation at 65 ℃ for 10min followed by incubation at 95 ℃ for 5 min.

A method for fluorescent quantitative PCR detection of an animal cell sample, comprising: the kit is used for pretreatment to obtain an amplification template, and primers are used for amplification.

The invention has simple preparation and easy preparation of the lysis solution under the condition of ensuring good stability of the lysis solution. The lysate included water, Tris-HCl, Tween, Triton X-100, and EDTA. The lysate can ensure that the nucleic acid can be extracted without adding proteinase K in the lysis process, shorten the period of extracting sample nucleic acid by the lysate, and reduce the cost of the lysate. The method for extracting nucleic acid is rapid and simple, low in cost and sensitive in detection, and the detection result of the genome extracted by the method is similar to that of the genome extracted by a commercial kit. The reagent is used for extracting the genomic DNA of the buccal swab, the nucleic acid extraction is completed within 20 minutes, and the fluorescent quantitative PCR detection is carried out by diluting a sample by a certain multiple.

Drawings

FIG. 1 shows fluorescence PCR amplification detection of 8 human oral samples from different sources;

FIGS. 2, 3 and 4 show the results of three dilutions and triplicate determinations of each sample.

Detailed Description

The invention provides the lysis solution and the application thereof, and the technical personnel can appropriately improve the technological parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1

A preparation for rapidly extracting genomic DNA lysate of an oral swab comprises the following steps:

components of a lysate: Tris-HCl 8.520 mM, 0.8% Tween-20, 0.1% Triton X-100, 10mM EDTA.

10ml of lysis solution was prepared:

(1) sucking 2ml of 1M Tris-HCl 8.5 solution by a liquid transfer machine, and adding the solution into a clean and sterile 15ml centrifuge tube;

(2) diluting Tween-20, namely, firstly diluting to 8% Tween-20, namely adding 80 mu l of Tween-20 into 910 mu l of ultrapure water, and reversing and uniformly mixing;

(3) sucking 1ml of 8% Tween-20 prepared in the step (2) by a pipette, and adding the Tween-20 into the 15m centrifugal tube in the step (1);

(4) diluting Triton X-100, diluting to 10% Triton X-100, adding 100 μ l Triton X-100 into 900 μ l ultrapure water, and mixing;

(5) continuously diluting the 10% Triton X-100 obtained in the step (4) to 1% TrionX-100, namely adding 100 μ l of 10% Tween-20 into 900 μ l of ultrapure water, and reversing and uniformly mixing;

(6) sucking 1ml of the 1% Triton X-100 prepared in the step (5) by a liquid transfer machine, and adding the sucked 1ml of the Triton X-100 into the 15m centrifugal tube in the step (1);

(7) pipette 200. mu.l of 0.5M EDTA solution.

(8) 5.8ml of ultrapure water is added into a 15ml centrifuge tube to make up to 10ml, and the mixture is inverted and mixed uniformly, so that the preparation of the lysate is finished.

Example 2

The method for quickly extracting the genomic DNA of the oral swab from the lysate comprises the following steps:

(1) putting the oral swab sample into a 1.5ml centrifuge tube;

(2) adding 200 mu l of nucleic acid lysate into the centrifugal tube in the step (1), and performing vortex oscillation;

(3) adding 4 mu l of 20mg/mL bovine serum albumin into the step (2), and performing instantaneous centrifugation after vortex oscillation;

(4) placing the centrifuge tube in a metal bath at 65 ℃ for incubation for 10 minutes;

(5) after instantaneous centrifugation, the centrifuge tubes were incubated for 5 minutes in a metal bath at 95 ℃;

(6) taking out the centrifuge tube, centrifuging the centrifuge tube in a centrifuge at normal temperature, and centrifuging the centrifuge tube for 5 minutes at 12000 r/min;

(5) absorbing the supernatant at the bottom of the centrifuge tube into a clean 1.5ml centrifuge tube to avoid absorbing the sediment;

(6) the supernatant is the extracted genome DNA, and the supernatant can be diluted by 5 to 10 times for qPCR.

Example 3

A fluorescent PCR amplification reaction solution used in a method for rapidly extracting a buccal swab genome for fluorescent PCR amplification comprises the concentrations or contents and components shown in the table 1.

TABLE 1

qPCR composition	Volume of
		template/Positive quality control/negative control	2μl
5×Multiplex qRT-PCR bufferⅡ	4μl
		Multi RT Enzyme MixⅡ	1μl
F Primer(10μM)	0.5μl
		R Primer(10μM)	0.5μl
P Primer(10μM)	0.4μl
		Sterilized water	Adding water to 20 μ l

Wherein:

f Primer sequence is ACCAGTAGCTGTTTCTGAACTCTTCAC

R Primer sequence is ATTGGTCTTGATTTTCCCTGTAC

P Primer sequence is AGATTTGGACCTGCGAGCG

The fluorescence PCR amplification reaction solution is used for performing fluorescence PCR amplification on RNAse in an oral cavity sample, and the method comprises the following steps: mu.L of the supernatant (example 2) was diluted 5 times, 10 times and 20 times, respectively, and added to the prepared fluorescent PCR reaction solution for amplification in a Berle fluorescent PCR apparatus.

The fluorescent PCR amplification procedure is as follows:

the first step is as follows: cycling at 95 ℃ for 3min for 1 time.

The second step is as follows: the fluorescence was collected at 95 ℃ for 15s and at 58 ℃ for 30s, and the cycle was repeated 40 times.

And (4) analyzing results: in the fluorescent quantitative PCR reaction of parallel amplification, negative control has no amplification, and positive quality control has amplification, which indicates that the reaction system is normal.

8 human oral samples with different sources are treated by the rapid extraction method, then the RNAse primers are used for carrying out fluorescence PCR amplification detection, the result is shown in figure 1, the background signals of all reaction holes are good, the Ct value is 28-36, the fluorescence amplification curve is in a typical S shape, and the method for rapidly extracting the oral swab sample for fluorescence PCR amplification provided by the embodiment 3 of the invention has good feasibility. 8 human blood samples with different sources are subjected to fluorescence PCR amplification detection, and each sample is diluted by three gradients: the samples were diluted 5-fold, 10-fold, and 20-fold, and the results are shown in fig. 2, 3, and 4 (each of which contains amplification curves of 8 samples).

TABLE 2

As can be seen from the figure and Table 2, the Ct values of the curves for the parallel experiments for each sample are very close as shown in Table 2 ("NA": indicating that the parallel experiment was run but no amplification data, "/": indicating that the parallel experiment was not run). The method for rapidly extracting the blood sample for the fluorescent PCR amplification provided by the embodiment of the invention has reliable repeatability and better uniformity.

Comparative example 1 comparative test on the Presence or absence of proteinase K addition

(1) Taking 5 parts of oral swab samples, putting the samples into a 1.5ml centrifuge tube, and adding 4 mul of proteinase K; another 5 oral swab samples were placed in 1.5ml centrifuge tubes without proteinase K.

(2) 200. mu.l of nucleic acid lysate was added to each of the above samples, followed by vortexing.

(3) Add 4. mu.l of 20mg/mL bovine serum albumin, vortex and shake, then perform an instantaneous centrifugation.

(4) The centrifuge tubes were incubated in a 65 ℃ metal bath for 10 minutes.

(5) After the transient centrifugation, the centrifuge tubes were incubated in a metal bath at 95 ℃ for 5 minutes.

(6) And taking out the centrifuge tube, centrifuging the centrifuge tube in a centrifuge at normal temperature, and centrifuging the centrifuge tube for 5 minutes at 12000 r/min.

(5) And (4) sucking the supernatant at the bottom of the centrifuge tube into a new 1.5ml centrifuge tube to avoid sucking the sediment.

(6) The supernatant is the extracted genomic DNA, and the supernatant is diluted by 5 times for qPCR.

(7) The qPCR detection gene is human RANseP, and the system is

qPCR composition	Volume of
		template/Positive quality control/negative control	2μl
5×Multiplex qRT-PCR bufferⅡ	4μl
		Multi RT Enzyme Mix Ⅱ	1μl
F Primer(10μM)	0.5μl
		R Primer(10μM)	0.5μl
P Primer(10μM)	0.4μl
		Sterilized water	Adding water to 20 μ l

(8) The conditions used for qPCR are

The first step is as follows: cycling at 95 ℃ for 3min for 1 time.

(9) The experimental results are summarized in the following table:

TABLE 3

Remarking: NTC represents negative quality control without template; N/A represents the CT-free value.

(10) And (4) conclusion: the CT values of the added proteinase K (CT mean value of 31.162) and the added proteinase K (CT mean value of 30.680) have no obvious difference, which shows that the lysate of the invention can obtain good lysis effect even if the proteinase K is not added.

Comparative example 2 comparison test with EDTA addition:

(1) 8 oral swab samples were taken and placed into a 1.5ml centrifuge tube, and another 8 oral swab samples were taken and placed into a 1.5ml centrifuge tube.

(2) Mu.l of the nucleic acid lysate containing 10mM EDTA was added to the first 8 samples, and 200. mu.l of the nucleic acid lysate containing no 10mM EDTA was added to the latter 8 samples, followed by vortexing and then performing transient centrifugation.

(3) The centrifuge tubes were incubated in a 65 ℃ metal bath for 10 minutes.

(4) After the transient centrifugation, the centrifuge tubes were incubated in a metal bath at 95 ℃ for 5 minutes.

(5) And taking out the centrifuge tube, centrifuging the centrifuge tube in a centrifuge at normal temperature, and centrifuging the centrifuge tube for 5 minutes at 12000 r/min.

(6) And (4) sucking the supernatant at the bottom of the centrifuge tube into a new 1.5ml centrifuge tube to avoid sucking the sediment.

(7) The supernatant is the extracted genomic DNA, and the supernatant is diluted by 5 times for qPCR.

(8) The qPCR detection gene is human RANseP, and the system is

(9) The conditions used for qPCR are

The first step is as follows: cycling at 95 ℃ for 3min for 1 time.

(10) The experimental results are summarized in the following table:

(10) And (4) conclusion: the CT values of the lysis solution containing 10mM EDTA (with the CT mean value of 31.029) and the lysis solution not containing 10mM EDTA (with the CT mean value of 34.217) are obviously different, so that the lysis solution contains 10mM EDTA in order to ensure the stability of the subsequent qPCR result.

Comparative example 3 comparative test of dilution or not of lysate:

(1) 8 mouth swab samples were placed into 1.5ml centrifuge tubes.

(4) The centrifuge tubes were incubated in a 65 ℃ metal bath for 10 minutes.

(6) The supernatant is the extracted genome DNA, and qPCR is respectively carried out after the supernatant is not diluted, is diluted by 5 times and is diluted by 10 times.

(7) The qPCR detection gene is human RANseP, and the system is

(8) The conditions used for qPCR are

The first step is as follows: cycling at 95 ℃ for 3min for 1 time.

(9) The experimental results are summarized in the following table:

(10) And (4) conclusion: undiluted (CT mean 32.855) gave poorer results than diluted 10-fold (CT mean 32.085) and 5-fold (30.326), so the dilution procedure was beneficial for qPCR, with 5-fold dilution giving better results than 10-fold dilution, so 5-fold dilution was chosen.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A lysis solution comprising water, Tris-HCl, Tween, Triton X-100 and EDTA.

2. Lysis solution according to claim 1, characterized in that it consists of water and of:

10-30 mM Tris-HCl, 0.5-1 wt% Tween, 0.01-1 wt% Triton X-100 and 1-20 mM EDTA.

3. Lysis solution according to claim 2, characterized in that it consists of water and of:

20mM Tris-HCl, 0.8 wt% Tween-20, 0.1 wt% Triton X-100 and 10mM EDTA.

4. Use of a lysate according to any one of claims 1 to 3 for lysing an animal cell sample.

5. The use of claim 4, wherein the animal cell sample is an animal buccal swab sample.

6. A method for extracting nucleic acid from an animal cell sample is characterized in that the sample to be extracted is mixed with the lysis solution of any one of claims 1 to 3, the mixture is mixed with a bovine serum albumin solution after vortex oscillation, the mixture is incubated for 8 to 12min at 60 to 70 ℃ after vortex oscillation again, the mixture is incubated for 4 to 6min at 90 to 100 ℃, and supernatant containing nucleic acid is obtained after centrifugation.

7. The method for extracting nucleic acid according to claim 6, wherein the sample to be extracted is a buccal swab, and 200. mu.L of the lysate is mixed with each buccal swab.

8. The method for extracting nucleic acid according to claim 6, wherein the concentration of bovine serum albumin in the bovine serum albumin solution is 20mg/mL, and the volume ratio of the bovine serum albumin solution to the nucleic acid lysate is 4: 200.

9. the method for extracting nucleic acid according to claim 6, wherein the incubation is performed at 60-70 ℃ before instantaneous centrifugation; and (3) performing instantaneous centrifugation before incubation at 90-100 ℃.

10. The method for extracting nucleic acid according to claim 6, wherein the centrifugation is performed at 12000 rpm for 5 minutes.