CN114672541A

CN114672541A - Nucleic acid release solution, kit and nucleic acid release method

Info

Publication number: CN114672541A
Application number: CN202210276947.XA
Authority: CN
Inventors: 赵斯斯; 薛茜
Original assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Current assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-06-28
Anticipated expiration: 2042-03-21
Also published as: CN114672541B

Abstract

The invention discloses a nucleic acid release solution, a kit and a nucleic acid release method, and relates to the technical field of biology. The cleaning agent comprises Tris-HCl, ammonium chloride, KOH, Tween-20 and water, and the concentration of each component is as follows: 40-60mM Tris-HCl, 100-200mM ammonium chloride, 30-50mM KOH, and Tween-20 accounting for 0.02-0.2% of the total volume of the nucleic acid release solution. When the nucleic acid release solution is used for treating a sample, specific steps of protein removal, RNA removal and secondary metabolite removal are not needed, organic solvent extraction is also not needed, the genome DNA with stable quality can be obtained, and the nucleic acid release solution can be directly used in the fields of PCR amplification, detection, identification and the like without subsequent purification steps. The nucleic acid release solution has the technical advantage of short time consumption in the operation process, and is more convenient for laboratory scientific research personnel or medical personnel to use.

Description

Nucleic acid release solution, kit and nucleic acid release method

Technical Field

The invention relates to the technical field of biology, and particularly relates to a nucleic acid release solution, a kit and a nucleic acid release method.

Background

Genomic DNA (gDNA) is the complete genetic information including coding and non-coding sequences in haploid cells. All life information can be found from the whole genome DNA, including the problems of gene expression, gene mutation and single nucleotide polymorphism which are closely related to diseases. Blood samples have remarkable reference value for clinical diagnosis and research, and hematology, genetics and biochemistry all need the blood samples to identify and judge various human diseases. Therefore, the isolation of genomic DNA from blood is the basis for clinical diagnosis and genomic research.

There are many PCR reaction inhibitors in whole blood, e.g., hemoglobin, proteins, salts, anticoagulants, etc., making it impossible to directly perform qPCR/RT-qPCR reactions using whole blood. Therefore, it is now clinically necessary to extract DNA from whole blood.

The traditional blood genome DNA extraction method mainly comprises the following steps: firstly, lysing erythrocytes with low permeability, then centrifugally collecting leukocytes, lysing the leukocytes by using cell lysate to release genome DNA, extracting by using an organic solvent (namely phenol/chloroform extraction) to remove impurities such as protein, heme and the like, and finally precipitating and dialyzing by using ethanol and the like to separate the DNA.

Currently, commercially available blood genomic DNA extraction kits usually release DNA from leukocytes using cell lysates, digest and decompose proteins and RNA sufficiently using proteinase K, ribonuclease, etc., and then separate genomic DNA by phenol/chloroform extraction or silica gel membrane chromatography. The methods can generally obtain DNA with high purity and less impurities, but the methods all require long incubation and precipitation, and have the problems of long and complicated operation steps, long waiting time and the like.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a nucleic acid release solution, a kit and a nucleic acid release method so as to realize the rapid release of nucleic acid, simplify the operation steps and shorten the processing time.

The invention is realized in the following way:

the invention provides a nucleic acid release solution, which comprises Tris-HCl, ammonium chloride, KOH, Tween-20 and water, wherein the concentration of each component is as follows: 40-60mM Tris-HCl, 100-200mM ammonium chloride, 30-50mM KOH, and Tween-20 accounting for 0.02-0.2% of the total volume of the nucleic acid releasing solution.

The inventors have creatively found that a nucleic acid releasing solution can be prepared by combining Tris-HCl, ammonium chloride, KOH and Tween-20 at the above concentrations. When the nucleic acid release solution is used for treating a sample, specific steps of protein removal, RNA removal and secondary metabolite removal are not needed, organic solvent extraction is also not needed, the genome DNA with stable quality can be obtained, and the nucleic acid release solution can be directly used in the fields of PCR amplification, detection, identification and the like without subsequent purification steps. The nucleic acid release liquid has the technical advantage of short time consumption in the operation process, is more convenient for laboratory scientific research personnel or medical personnel to use, and greatly reduces the workload of the scientific research personnel or the medical personnel. In addition, the toxic reagent used in the operation process is few and is safe.

After the sample is treated by the nucleic acid release solution, the released nucleic acid can meet the application requirements of methods such as PCR, qPCR and the like.

In the nucleic acid release solution, Tris-HCl can provide proper pH buffer conditions for nucleic acid release, and is helpful for better release of nucleic acid; ammonium chloride is a main effect substance of the erythrocyte lysate, and ammonia ions can not pass through cell membranes, but other ions can pass through the cell membranes, so that the concentration difference of ions inside and outside the cells is caused, the osmotic pressure difference is formed, external water can be diffused into the cells, the erythrocytes are expanded, and the lysis effect is achieved; KOH provides ions which can play a role in adjusting pH; tween-20 is a nonionic surfactant, has hemolytic effect, can cause in vitro hemolysis and erythrocyte morphology change, and can reduce viscosity of reaction system, so that blood precipitate treated by nucleic acid release solution does not stick to tube wall.

Since red blood cells in whole blood are anucleated, the release of nucleic acids requires the removal of DNA-free anucleated red blood cells in order not to affect leukocyte lysis and DNA release. Through the synergistic effect of the four substances (Tris-HCl, ammonium chloride, KOH and Tween-20) provided by the invention, erythrocytes can be subjected to hemolytic lysis, and the interference of erythrocytes on the release of nucleic acid is avoided; at the same time, the leucocytes can be lysed to release the nucleic acid.

The inventor finds that if the Tween-20 is replaced by TritonX-100, the expected effect cannot be achieved, and an ideal qPCR detection result cannot be obtained. In addition, the inventors also found that the nucleic acid release solution containing proteinase K had poor nucleic acid release effect and the qPCR detection result was not satisfactory. It should be noted that the reaction solution after the treatment of the nucleic acid release solution provided by the present invention can be directly coupled and compatible with downstream PCR experiments, and does not need to purify the nucleic acid. Has the technical advantage of sample direct expansion.

It should be noted that, after a large number of experiments, the inventor has verified that the hemolytic property is reduced compared with tween-20 after adding tween-80 as a substitute for tween-20, and a large number of screening tests show that tween-20 has the best hemolytic property.

If ammonium chloride is replaced by ammonium nitrate and the like, on one hand, the effect of releasing nucleic acid can be influenced, and on the other hand, incompatibility with a subsequent PCR enzymatic reaction system can be caused, so that the subsequent PCR reaction is inhibited.

If KOH is replaced by NaOH, the subsequent PCR enzymatic reaction system can be inhibited due to the strong alkalinity of the NaOH.

The nucleic acid extracting solution can be used for developing a nucleic acid releasing solution of the blood direct amplification probe method qPCR without nucleic acid extraction.

In an alternative embodiment, the concentration of Tris-HCl is 40mM, 45mM, 50mM, 55mM or 60 mM.

In a preferred embodiment of the present invention, the pH of Tris-HCl is 7.5-9.0. For example: 7.5, 8.0, 8.5 or 9.0. The concentration of ammonium chloride is 100mM, 130mM, 150mM, 180mM or 200 mM. The concentration of KOH was 30mM, 32mM, 34mM, 35mM, 36mM, 38mM or 40 mM. Tween-20 accounts for 0.02%, 0.05%, 0.06%, 0.08%, 0.1%, 0.15% or 0.2% of the total volume of the nucleic acid releasing solution. Under the above ratio of the components, the nucleic acid releasing solution can release nucleic acid in a target sample well.

In a preferred embodiment of the present invention, the concentrations of the above components are as follows: 50-60mM Tris-HCl, 150-200mM ammonium chloride, 35-50mM KOH, and Tween-20 accounting for 0.05-0.2% of the total volume of the nucleic acid release solution.

In a preferred embodiment of the present invention, the concentrations of the above components are as follows: 50mM Tris-HCl, 200mM ammonium chloride, 35mM KOH, and Tween-20 accounting for 0.05 percent of the total volume of the nucleic acid release solution.

The present invention also provides a reagent or kit comprising: the nucleic acid releasing solution.

In one embodiment, the kit further comprises a qPCR or PCR mix reaction solution.

The form of the above reagent includes, but is not limited to: lyophilized formulations and liquid formulations. When a lyophilized preparation is prepared, it can be diluted with water for use.

The present invention also provides a method for nucleic acid release, comprising: mixing a biological sample to be released with the nucleic acid releasing solution.

After mixing, under the synergistic effect of the four substances, the cell can achieve the effect of cracking, so that the nucleic acid molecules are released.

In a preferred embodiment of the present invention, the mixing volume ratio of the biological sample to be released with nucleic acid to the nucleic acid releasing solution is 0.5-2: 1.

The mixing volume can enable the nucleic acid in the biological sample to have a better release effect.

In an alternative embodiment, after the mixing, vortex mixing followed by transient (or short) centrifugation may be selected to ensure sufficient mixing.

In a preferred embodiment of the present invention, the mixing further comprises placing the container containing the mixture of the biological sample to be released and the nucleic acid releasing liquid at 90-100 ℃ for reaction for 5-10 min;

preferably, the reaction is carried out at 95 ℃ for 5-10 min. Through reaction at 95 ℃, protein, cell membranes, impurities and the like can better aggregate and precipitate, and released nucleic acid is dissociated in supernatant; in addition, heating can also facilitate continued pyrolysis.

In a preferred embodiment of the present invention, after the reaction, the step of centrifuging the reaction solution after the reaction is further included.

In an alternative embodiment, the centrifuge rotation speed is 9000-. Centrifuging for 1-5 min; for example, 2 min. Impurities can be well removed through centrifugation, and nucleic acid substances in the supernatant are reserved for subsequent amplification and other applications.

Further, in the qPCR or PCR reaction, the supernatant obtained by centrifugation (i.e. the template) is added in an amount of less than 40%, e.g. 10% -30% of the total reaction system (e.g. qPCR). In an alternative embodiment, the template is added in an amount of 20% of the reaction system.

In a preferred embodiment of the present invention, the biological samples to be released include, but are not limited to: blood, serum, plasma, or urine.

The invention has the following beneficial effects:

the invention creatively discovers that a nucleic acid release solution can be prepared by combining Tris-HCl, ammonium chloride, KOH and Tween-20 with specific concentrations. When the nucleic acid release solution is used for treating a sample, specific steps of protein removal, RNA removal and secondary metabolite removal are not needed, organic solvent extraction is also not needed, the genome DNA with stable quality can be obtained, and the nucleic acid release solution can be directly used in the fields of PCR amplification, detection, identification and the like without subsequent purification steps. The nucleic acid release liquid has the technical advantage of short time consumption in the operation process, is more convenient for laboratory scientific research personnel or medical personnel to use, and greatly reduces the workload of the scientific research personnel or the medical personnel. In addition, the toxic reagent used in the operation process is few and is safe.

Specifically, Tris-HCl in the nucleic acid release solution can provide proper pH buffer conditions for nucleic acid release, and is beneficial to better release of nucleic acid; ammonium chloride is a main effect substance of the erythrocyte lysate, and ammonia ions can not pass through cell membranes, but other ions can pass through the cell membranes, so that the concentration difference of ions inside and outside the cells is caused, the osmotic pressure difference is formed, external water can be diffused into the cells, the erythrocytes are expanded, and the lysis effect is achieved; KOH provides ions which can play a role in adjusting pH; tween-20 is a nonionic surfactant, has hemolytic effect, can cause in vitro hemolysis and erythrocyte morphology change, and can reduce viscosity of reaction system, so that blood precipitate treated by nucleic acid release solution does not stick to tube wall.

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 will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph showing the amplification curves of the control group and the experimental group in the experiment example 1, in which the primer A is used as the verification primer to perform qPCR.

FIG. 2 is a graph showing the amplification curves of the control group and the experimental group in the qPCR with the primer B as the verification primer in the experimental example 1.

FIG. 3 is a graph showing the amplification curves of the control group and the experimental group in the qPCR with the C primer as the verification primer in the experimental example 1.

FIG. 4 is a graph showing the amplification curves of the control group and the experimental group in the qPCR with the D primer as the verification primer in the experimental example 1.

FIG. 5 is a graph showing the amplification curves of example 1, comparative example 2 and comparative example 3 in which the primer B in Experimental example 2 was used as a verification primer for qPCR.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a nucleic acid release solution and a release method (or a using method) for a blood direct amplification probe method qPCR.

Wherein the nucleic acid releasing solution contains: 50mM Tris-HCl (pH8.0), 35mM potassium hydroxide, 200mM ammonium chloride, 0.05% Tween-20 (volume percent concentration).

The using method comprises the following steps:

s1, selecting a clean 200. mu.l EP tube, and adding 50. mu.l of nucleic acid releasing solution to the EP tube.

S2, adding a blood sample (the blood source used in the invention is the company) with the volume equal to that of the nucleic acid release liquid into the EP tube, uniformly mixing by vortex, and centrifuging for a short time.

And S3, placing the reaction solution in a PCR instrument, reacting for 5min at 95 ℃, placing the reaction solution in a centrifuge for centrifugation for 2min, and taking the supernatant to be directly used as a qPCR reaction template.

Further, in step S3, the centrifugal speed of the centrifuge is 10000 r/min. The amount of the template added in step S3 was 20% of the reaction system.

Example 2

Wherein the nucleic acid releasing solution contains: 50mM Tris-HCl (pH8.0), 35mM potassium hydroxide, 200mM ammonium chloride, 0.2% Tween-20 (volume percent concentration).

The using method comprises the following steps:

s1, a clean 200. mu.l EP tube was selected, and 50. mu.l of the nucleic acid releasing solution was added to the EP tube.

S2, adding a blood sample with a volume equal to that of the nucleic acid release solution into the EP tube, uniformly mixing by vortex, and centrifuging for a short time.

Further, in step S3, the centrifuge rotation speed of the centrifuge is 10000 r/min. The amount of the template added in step S3 was 20% of the reaction system.

Example 3

The embodiment provides a method for detecting qPCR by a blood direct amplification probe method, which comprises the following steps:

further, the probe method qPCR Mix adopts 2 XTaqMan Fast qPCR Master Mix of Shanghai Productus No. B639274.

S4, configuring a probe method qPCR reaction system as shown in the following table 1:

table 1 qPCR reaction system.

S5, probe qPCR reaction program see table 2:

TABLE 2 qPCR reaction procedure

Further, ROX used mainly comprises two concentrations, ROX Reference Dye (L) and ROX Reference Dye (H), selected according to different types of quantitative fluorescence PCR instruments.

Further, in step S4, the ROX Reference Dye needs to be selected according to the model of the device: the ROX Reference Dye (H) is suitable for being in the ABI Prism7000/7300/7700/7900, Eppendorf Realplex 4, ABI Step One and ABI Step One Plus types; suitable models of the ROX Reference Dye (L) are ABI Prism7500, ABI Prism7500 Fast, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000; the models that do not require ROX correction are Roche LightCycler480, Roche Light Cycler96, MJ Research Chromo4, Opticon (II), Bio-Rad iCycler iQ, iQ5, Bio-Rad CFX96, Corbett Rotor Gene 6000, Eppendorf Realplex 2, and so on.

Further, the fluorescent quantitative PCR instruments used in this example were all Tianlong Gentier 96R.

Comparative example 1

The comparative example provides a nucleic acid release solution and a method for qPCR (quantitative polymerase chain reaction) by a blood direct amplification probe method.

Wherein, the nucleic acid release solution: 50mM Tris-HCl (pH8.0), 35mM potassium hydroxide, 200mM ammonium chloride, 0.05% TritonX-100.

The using method comprises the following steps:

S3, placing the reaction solution in a PCR instrument, reacting for 5-10min at 95 ℃, placing in a centrifuge for centrifuging for 2min, and taking the supernatant to be directly used as a qPCR reaction template.

Further, in step S3, the centrifugal speed of the centrifuge is 10000 r/min.

The amount of the template added in step S3 was 20% of the reaction system.

Comparative example 2

The comparative example provides a nucleic acid release solution and a method for qPCR by a blood direct amplification probe method.

Wherein, the nucleic acid releasing solution: 50mM Tris-HCl (pH8.0), 35mM potassium hydroxide, 200mM ammonium chloride, 0.05mg/ml proteinase K.

The using method comprises the following steps:

Comparative example 3

Wherein, the nucleic acid releasing solution: 50mM Tris-HCl (pH8.0), 35mM sodium hydroxide, 200mM magnesium chloride, 0.05% Tween-20.

The using method comprises the following steps:

Further, in step S3, the centrifugal speed of the centrifuge is 10000 r/min.

The amount of the template added in step S3 was 20% of the reaction system.

Experimental example 1

In order to verify the amplification effect (qPCR detection result) of the nucleic acid releasing solution provided in example 1 of the present invention, the effect of the nucleic acid releasing solution used in the direct blood amplification probe method qPCR in example 1 (experimental group) was compared with the effect of the nucleic acid releasing solution commercially available from the control kit (control group selected from nucleic acid releasing solutions manufactured by suzhou corporation).

The control group and the experimental group were verified by the direct blood amplification probe method qPCR detection method in example 3. The total system volume is 20 mul, the template volume is 4 mul, and the probe volume is 0.4 mul. Two parallels were set for each control group and experimental group, and the average was taken. Four validation primers A, B, C, D were selected, the sequences of which are shown in table 3 below:

TABLE 3 sequence information Table

The results of comparing the primer A control group with the experimental group are shown in Table 4 below. FIG. 1 shows the corresponding amplification curves, wherein line 1 is the experimental group and line 2 is the control group.

TABLE 4A comparison of primer control groups with experimental groups

The results of comparing the primer B control group with the experimental group are shown in Table 5 below. FIG. 2 shows the corresponding amplification curves, wherein line 1 is the experimental group and line 2 is the control group.

TABLE 5B comparison of primer control groups with experimental groups

The results of comparing the C primer control group with the experimental group are shown in Table 6 below. FIG. 3 shows the corresponding amplification curves, wherein line 1 is the experimental group and line 2 is the control group.

TABLE 6C comparison of primer control groups with experimental groups

The results of comparing the primer D control group with the experimental group are shown in Table 7 below. FIG. 4 shows the corresponding amplification curves, wherein line 1 is the experimental group and line 2 is the control group.

TABLE 7 comparison of primer control group to Experimental group

The analysis results prove that the kit can directly carry out qPCR amplification reaction after simply pretreating blood, and can obtain an ideal qPCR detection result.

Experimental example 2

In this experimental example, the amplification effect of the nucleic acid releasing solution was compared in example 1 and comparative examples 1 to 3, respectively.

The blood direct amplification probe method qPCR detection method in example 3 was used for verification. The total system was 20. mu.l, the template amount was 4. mu.l, and the probe amount was 0.4. mu.l. Each group of two replicates was averaged. Among them, primers B shown in Table 3 were selected as verification primers.

The following Table 9 shows the comparative results of the primers B, example 1 and comparative examples 1 to 3. FIG. 5 is a corresponding amplification curve, where line 1 is the results of example 1, line 2 is the results of comparative example 1, line 3 is the results of comparative example 2, and line 4 is the results of comparative example 3.

Table 9 is a statistical table of comparative results of B primer example 1 and comparative examples 1-3.

The analysis results prove that the kit can directly carry out qPCR amplification reaction after simply pretreating blood, the effect of the embodiment 1 is optimal, an ideal qPCR detection result can be obtained, and the amplification result can be influenced by replacing Tween-20 with TritonX-100 or proteinase K, so that the amplification is not ideal (the comparative example 1-2); the substitution of ammonium chloride for magnesium chloride also severely affected the amplification results, leading to an unsatisfactory amplification (comparative example 3).

It can be seen that the components in the present invention synergistically exert their unique effects. Tris-HCl in the nucleic acid release solution can provide proper pH buffer conditions for nucleic acid release, and is helpful for better release of nucleic acid; ammonium chloride is a main effect substance of erythrocyte lysate, and ammonia ions cannot pass through cell membranes, but other ions can pass through the cell membranes, so that the concentration difference of ions inside and outside the cells is caused, osmotic pressure difference is formed, external water can be diffused into the cells, the erythrocytes are expanded, and the lysis effect is achieved; KOH provides ions which can play a role in adjusting pH; tween-20 is a nonionic surfactant, has hemolytic effect, can cause in vitro hemolysis and erythrocyte morphology change, and can reduce viscosity of reaction system, so that blood precipitate treated by nucleic acid release solution does not stick to tube wall.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

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Claims

1. The nucleic acid release solution is characterized by comprising Tris-HCl, ammonium chloride, KOH, Tween-20 and water, wherein the concentration of each component is as follows: 40-60mM Tris-HCl, 100-200mM ammonium chloride and 30-50mM KOH, wherein the Tween-20 accounts for 0.02-0.2 percent of the total volume of the nucleic acid release solution.

2. The nucleic acid releasing solution according to claim 1, wherein Tris-HCl has a pH of 7.5 to 9.0.

3. The nucleic acid releasing solution according to claim 1 or 2, wherein the concentrations of the respective components are as follows: 50-60mM Tris-HCl, 150-200mM ammonium chloride and 35-50mM KOH, wherein the Tween-20 accounts for 0.05-0.2 percent of the total volume of the nucleic acid release solution.

4. The nucleic acid releasing solution according to claim 3, wherein the concentrations of the respective components are as follows: 50mM Tris-HCl, 200mM ammonium chloride, 35mM KOH, and the Tween-20 accounts for 0.05 percent of the total volume of the nucleic acid release solution.

5. A reagent or kit, characterized in that it comprises: the nucleic acid releasing solution according to any one of claims 1 to 4.

6. A method for nucleic acid delivery, comprising: mixing a biological sample from which nucleic acids are to be released with the nucleic acid releasing solution according to any one of claims 1 to 4.

7. The method according to claim 6, wherein the mixing volume ratio of the biological sample to be released with nucleic acid to the nucleic acid releasing solution is 0.5 to 2: 1.

8. The method according to claim 6 or 7, wherein the mixing further comprises reacting the container containing the mixture of the biological sample to be released and the nucleic acid releasing solution at 90-100 ℃ for 5-10 min;

preferably, the reaction is carried out at 95 ℃ for 5 to 10 min.

9. The method according to claim 8, further comprising centrifuging the reacted reaction solution after the reaction.

10. The method according to claim 6, wherein the biological sample from which nucleic acids are to be released is selected from the group consisting of blood, serum, plasma, and urine.