CN113817718B - Method for lysing cells by using eutectic solvent and directly using the same in polymerase chain amplification reaction - Google Patents
Method for lysing cells by using eutectic solvent and directly using the same in polymerase chain amplification reaction Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a method for lysing cells by using eutectic solvent and directly using the cells in polymerase chain reaction, which comprises the following steps: firstly preparing a eutectic solvent, then adding a trace amount of biological detection material into the eutectic solvent, incubating at 50-60 ℃, and vibrating for 10-20 minutes, then directly adding the trace amount of the eutectic solvent into a PCR amplification system, wherein the quantity of the eutectic solvent is not more than 30% of the total amplification volume, and the mass ratio of the detection material to the ionic liquid is 1:99-1:50. The method for extracting DNA by using the eutectic solvent to lyse cells has the characteristics of simple operation, short time consumption, low cost and high extraction efficiency, widens the application field of the eutectic solvent, can carry out PCR detection by using a trace or trace amount of lysate of a biological sample, and is equivalent to or slightly superior to a magnetic bead reagent lysis purification method under certain conditions.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for lysing cells by using a eutectic solvent and directly using the cells in a polymerase chain amplification reaction.
Background
DNA is an important genetic information material of organisms, and detection and analysis thereof are widely applied to various fields, and particularly, the most widely applied polymerase chain amplification (PCR) has become an indispensable technical means in clinical disease diagnosis, food safety, environmental management and animal epidemic disease, especially forensic identification.
Biological samples in the criminal investigation field have their specificity, and samples collected on site are often trace, even trace. Although the PCR reaction can be completed theoretically by one DNA template, a certain amount of DNA template number and good quality are required for the PCR process to be completed in practice. Particularly, STR typing technology is often used in the forensic identification process, the STR typing technology is greatly influenced by the number and quality of DNA templates, and if the template quantity is insufficient, the locus is lost or the peak value is too low, so that the identification accuracy cannot be ensured. At present, the DNA in the biological sample needs to be extracted and purified before PCR is performed, and the main methods include Chelex-100 method, organic method (saturated phenol method), silica method (silica bead method, magnetic bead method), salting-out method, and the like, although each has advantages, the methods are not satisfactory for DNA in trace or even trace biological samples, because the methods all need multiple steps of operations including cracking, adsorption, rinsing and final elution to obtain cleaner DNA, and some DNA is inevitably lost in the processes, and finally the template requirement of PCR is hardly met. Therefore, in recent years, the lysate direct PCR amplification technology is gaining attention, because no complicated operation process is needed, the loss of DNA in trace biological samples is avoided, and the lysate direct PCR amplification technology is particularly suitable for trace or trace biological samples, especially contact samples, such as DNA in fingerprints. To date, there are several patents related to this technology, for example, chinese patent (CN 105441421A) "a cell lysis method", the main functional component of which is proteinase K, mainly suitable for the lysis of cultured cell suspensions. Chinese patent (CN 102177250A) 'method for direct amplification from crude nucleic acid samples', which uses sodium hydroxide as the cell lysis component, the crude nucleic acid sample produced by this method is only suitable for the specific PCR direct buffer of this patent, which contains a certain amount of non-ionic surfactant, glycerol and Bovine Serum Albumin (BSA). These compounds are not contained in conventional commercial PCR buffers and therefore may affect the amplification of crude nucleic acid samples after sodium hydroxide cleavage in conventional commercial PCR buffers. In view of this, there is an urgent need for new solvents with good cell degradation and DNA extraction capabilities, while having no impact on the downstream PCR process.
The eutectic solvent (Deep eutectic solvent, DES) is used as a novel green solvent, and has the advantages of low-cost and abundant raw material sources, simple synthesis, no need of purification of products and the like, and is widely used for purification of proteins and nucleic acids in combination with other separation technologies, so that the novel solvent which has many similarities with ionic liquids is more environment-friendly and has degradability than the ionic liquids, and therefore attracts more and more attention. The eutectic solvent may be prepared simply by mixing a Lewis acid with a base, or a hydrogen bond donor (hydrogen bond donor) with a hydrogen bond acceptor (hydrogen bond acceptor). Most importantly, the nature of the solvent is tunable as desired. The prepared eutectic solvent has a melting point lower than that of the component compound, and exists in a liquid state at room temperature, even though the component compound is in a solid state at room temperature. Imidazole is a five-membered aromatic heterocyclic compound, and the molecular structure of the imidazole contains two meta-position nitrogen atoms. Imidazole is acidic and basic, and in an environment with pH <6, the N atom at the 3 position of the imidazole is protonated, so that the imidazole ring has positive charges, and when the imidazole ring is deprotonated under the condition of pH >6, an uncharged imidazole ring is formed, and the binding capacity of the imidazole ring to DNA can be used as a transport carrier of DNA.
Disclosure of Invention
The present invention is directed to a method for lysing cells using a eutectic solvent and directly using the same in a polymerase chain reaction, which solves the above-mentioned problems of the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for lysing cells using a eutectic solvent and directly for polymerase chain amplification reaction, comprising the steps of:
firstly preparing a eutectic solvent, then adding a trace amount of biological detection material into the eutectic solvent, incubating at 50-60 ℃, and vibrating for 10-20 minutes, then directly adding the trace amount of the eutectic solvent into a PCR amplification system, wherein the quantity of the eutectic solvent is not more than 30% of the total amplification volume, and the mass ratio of the detection material to the ionic liquid is 1:99-1:50.
As still further aspects of the invention: the eutectic solvent consists of imidazole and alkylated carboxylic acid, wherein the imidazole in the eutectic solvent exists in a positively charged state, and the alkylated carboxylic acid has the characteristic of dissolving cell membrane lipid, so that released DNA can be specifically combined with the imidazole. After the addition of the eutectic solvent to the PCR system, the pH of the reaction buffer of PCR is usually 8-9, in which the imidazole undergoes deprotonation, resulting in the disappearance of its electrostatic interaction with DNA, the detachment of DNA from imidazole, and the PCR reaction.
As still further aspects of the invention: imidazole to alkylated carboxylic acid molar ratio 1:2-2:1, under the airtight condition, stirring strongly at 60-80 ℃ until the solution becomes clear and transparent, adding water which is not more than 20% of the total volume into the system, and uniformly mixing.
As still further aspects of the invention: the mass ratio of the trace biological detection material to the eutectic solvent is preferably 1:90, and the volume of the eutectic solvent added into the PCR system is preferably 20% of the PCR volume.
As still further aspects of the invention: the molar ratio of imidazole to alkylated carboxylic acid is preferably 1:2 to 1:1, and the water addition is preferably 10%. The eutectic solvent has low melting point and low viscosity.
Compared with the prior art, the invention has the beneficial effects that:
the method for extracting DNA by using the eutectic solvent to lyse cells has the characteristics of simple operation, short time consumption, low cost and high extraction efficiency, widens the application field of the eutectic solvent, can carry out PCR detection by using a trace or trace amount of lysate of a biological sample, and is equivalent to or slightly superior to a magnetic bead reagent lysis purification method under certain conditions.
Drawings
FIG. 1 is a 500-fold diluted blood STR profile of imidazole/hexanoic acid eutectic solvent cleavage;
FIG. 2 is a STR profile of a 500-fold diluted blood with a magnetic bead extraction reagent.
Description of the embodiments
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to FIGS. 1-2, in one embodiment of the present invention, a method of lysing cells using a eutectic solvent and directly using the same for a polymerase chain amplification reaction is provided.
Examples
Preparing a eutectic solvent: firstly, accurately weighing 6.8 g of imidazole, then adding the imidazole into a beaker filled with 23.2 g of caproic acid, then stirring the mixture to form clear and transparent liquid at the temperature of 80 ℃ by using a magnetic stirrer, and then adding 3.5 ml of deionized water into the mixed liquid, and stirring the mixture for 10 minutes for later use.
Diluting human blood 500 times with physiological saline, taking 1 microliter of the diluent, dripping on a glass slide, naturally air-drying, wiping the blood spots with a cotton swab or a flocking swab, partially shearing the wiped cotton swab, loading the cotton swab into a 2.0 milliliter centrifuge tube, adding 100 microliter of imidazole/caproic acid eutectic solvent to completely submerge the sample, heating and incubating at 50 ℃ for shaking for 20 minutes, absorbing 2 microliters of lysate, directly adding the lysate into an STR-PCR system for amplification, and amplifying the total volume of 10 microliters. Meanwhile, 1 microliter of diluted blood is used for DNA magnetic bead reagent cracking purification, and 2 microliter of eluent is taken for amplification in an STR-PCR system after the steps of cracking, adsorption, rinsing and elution, and the total volume of amplification is 10 microliters.
Analysis of 50 samples of the two cleavage methods respectively shows that 33 cases of more than 12 loci are detected in STR typing patterns by using a magnetic bead reagent cleavage purification method, 16 loci are reached, and the detection rate for effective individual identification is 68%. 50. For example, 32 cases of over 12 loci are detected by STR typing detection by using imidazole/caproic acid eutectic solvent, the detection rate for effective individual identification is 64% when the number of loci is 16, and the peak value of a sample map is in the range of 200-1000 RFU.
Examples
Preparing a eutectic solvent: firstly, 13.6 g of imidazole is accurately weighed, then the imidazole is added into a beaker filled with 28.8 g of octanoic acid, the mixture is stirred on a magnetic stirrer at 60 ℃ until clear and transparent liquid is formed, and then 9.0 ml of deionized water is added into the system, and the mixture is stirred for 10 minutes for later use.
Diluting human blood 500 times with physiological saline, dripping 1 microliter of the diluent on a glass slide, naturally air-drying, sticking the blood spots by using transparent adhesive tape, partially shearing the stuck adhesive tape, loading the adhesive tape into a 2.0 milliliter centrifuge tube, adding 100 microliter of imidazole/octanoic acid eutectic solvent, completely immersing the sample, heating at 60 ℃ for incubation and shaking for 10 minutes, sucking 3 microliters of lysate, directly adding the lysate into an STR-PCR system for amplification, and amplifying the total volume of 10 microliters. Meanwhile, 1 microliter of diluted blood is taken for DNA magnetic bead reagent pyrolysis and purification, 3 microliter of eluent is taken for amplification in an STR-PCR system after the steps of pyrolysis, adsorption, rinsing and elution, and the total volume of amplification is 10 microliters.
Analysis of 50 samples of the two cleavage methods respectively shows that more than 12 loci are detected in the STR typing map by using a magnetic bead reagent cleavage purification method, and the detection rate of the effective individual identification is 80% when the number of loci is 16. More than 12 loci are detected in 50 cases by STR typing detection using imidazole/octanoic acid eutectic solvent, the detection rate for effective individual identification is 76% when more than 16 loci are detected, and the peak value of the sample map is in the range of 400-1600 RFU.
Examples
Preparing a eutectic solvent: firstly, 136 g of imidazole is accurately weighed, then the imidazole is added into a beaker filled with 88 g of butyric acid, the mixture is stirred on a magnetic stirrer at 70 ℃ until clear and transparent liquid is formed, then 30 ml of deionized water is added into the system, and the mixture is stirred for 10 minutes for later use.
Diluting human blood 500 times with physiological saline, dripping 1 microliter of the diluent on a glass slide, naturally air-drying, sticking the blood spots by using transparent adhesive tape, partially shearing the stuck adhesive tape, loading the adhesive tape into a 2.0 milliliter centrifuge tube, adding 100 microliter of imidazole/butyric acid eutectic solvent to completely submerge the sample, heating and incubating at the temperature of 55 ℃ for shaking for 15 minutes, sucking 1 microliter of lysate, directly adding the lysate into an STR-PCR system for amplification, and amplifying the total volume of 10 microliters. Meanwhile, 1 microliter of diluted blood is taken for DNA magnetic bead reagent pyrolysis and purification, and 1 microliter of eluent is taken for amplification in an STR-PCR system after the steps of pyrolysis, adsorption, rinsing and elution, and the total volume of amplification is 10 microliters.
Through the analysis of 50 samples of the two cracking methods respectively, more than 12 loci are detected in the STR parting map of the magnetic bead reagent cracking purification method, and the detection rate of the effective individual identification is 63% when the number of loci is 16. More than 12 loci are detected in 50 cases by STR typing detection using imidazole/butyric acid eutectic solvent, the detection rate for effective individual identification is 64% when more than 16 loci are detected, and the peak value of the sample map is in the range of 200-800 RFU.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation. The term "comprising" an element defined by the term "comprising" does not exclude the presence of other identical elements in a process, method, article or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A method for lysing cells using a eutectic solvent and directly for use in a polymerase chain amplification reaction, comprising the steps of:
firstly, preparing a eutectic solvent, then adding a trace amount of biological detection material into the eutectic solvent, incubating at 50-60 ℃, and vibrating for 10-20 minutes, then directly adding the trace amount of the eutectic solvent into a PCR amplification system, wherein the quantity of the eutectic solvent is not more than 30% of the total amplification volume, and the mass ratio of the detection material to the ionic liquid is 1:99-1:50;
the eutectic solvent consists of imidazole, alkylated carboxylic acid and water, wherein the imidazole in the eutectic solvent exists in a positively charged state, the alkylated carboxylic acid has the characteristic of dissolving cell membrane lipid, released DNA can be specifically combined with the imidazole, and after the eutectic solvent is added into a PCR system, the imidazole is deprotonated in the system due to the pH value of a reaction buffer solution of PCR is usually 8-9, so that electrostatic interaction between the imidazole and the DNA is eliminated, the DNA and the imidazole are separated, and PCR reaction is carried out.
2. A method for lysing cells using a eutectic solvent and directly for use in a polymerase chain amplification reaction according to claim 1 wherein the eutectic solvent is prepared by reacting imidazole with alkylated carboxylic acid in a molar ratio of 1:2-2:1, under the airtight condition, stirring strongly at 60-80 ℃ until the solution becomes clear and transparent, adding water which is not more than 20% of the total volume into the system, and uniformly mixing.
3. The method for directly performing Polymerase Chain Reaction (PCR) by using eutectic solvent to lyse cells according to claim 1, wherein the mass ratio of trace amount of biological detection material to eutectic solvent is 1:90, and the volume of eutectic solvent added into PCR system is 20% of PCR volume.
4. A method according to claim 2 for lysing cells using a eutectic solvent with a low melting point and a low viscosity and directly for use in a polymerase chain amplification reaction, wherein the molar ratio of imidazole to alkylated carboxylic acid is 1:2 to 1:1 and water is added in an amount of 10%.
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