CN113817718A - Cell lysate directly used for polymerase chain amplification reaction - Google Patents
Cell lysate directly used for polymerase chain amplification reaction Download PDFInfo
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- CN113817718A CN113817718A CN202111084649.2A CN202111084649A CN113817718A CN 113817718 A CN113817718 A CN 113817718A CN 202111084649 A CN202111084649 A CN 202111084649A CN 113817718 A CN113817718 A CN 113817718A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a cell lysate directly used for polymerase chain amplification reaction, which is characterized by comprising the following steps: firstly, preparing a eutectic solvent, then adding a trace biological detection material into the eutectic solvent, incubating at the temperature of 50-60 ℃, shaking for 10-20 minutes, and directly adding the trace eutectic solvent into a PCR amplification system, wherein the amount 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 cracking cells by using the eutectic solvent 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 trace or trace lysate of biological samples, and is equivalent to or slightly superior to a magnetic bead reagent cracking and purifying method under certain conditions.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cell lysate directly used for polymerase chain amplification reaction.
Background
DNA is used as an important genetic information substance of organisms, the detection and analysis of the DNA are widely applied to various fields, and particularly, the most widely applied polymerase chain amplification reaction (PCR) is an indispensable technical means in clinical disease diagnosis, food safety, environmental management and animal epidemic diseases, particularly in court identification.
Biological samples in the criminal investigation field have their particularity, and samples collected on site are often in trace amounts, even trace amounts. Although one DNA template can complete PCR reaction theoretically, a certain amount of DNA template and good quality are needed in practical work to complete the PCR process. Particularly, STR typing technology is frequently used in the forensic identification process, the influence of the number and quality of DNA templates is large, and if the amount of the templates is insufficient, the loss of loci or too low peak value can be caused, so that the identification accuracy cannot be ensured. At present, before PCR is carried out, DNA in a biological sample needs to be extracted and purified, and the main methods comprise a Chelex-100 method, an organic method (saturated phenol method), a silica method (silica bead method, magnetic bead method), a salting-out method and the like, and although the methods have advantages, the methods are not satisfactory for DNA in trace or even trace biological samples, because the methods all need multiple steps of operation, including cracking, adsorption, rinsing and final elution to obtain cleaner DNA, some DNA is inevitably lost in the operation processes, and finally the template requirements of PCR are difficult to meet. Therefore, in recent years, the direct PCR amplification technology of lysate has been gaining attention because it does not require complicated procedures, avoids the loss of DNA in trace amount of biological samples, and is particularly suitable for trace amount or trace amount of biological samples, especially for contact samples such as DNA in fingerprints. To date, several patents have been referred to, for example, Chinese patent (CN 105441421A) "a cell lysis method" whose main functional component is proteinase K, which is mainly suitable for the lysis of cultured cell suspensions. Chinese patent (CN 102177250 a) "method for direct amplification from crude nucleic acid sample" using sodium hydroxide as a cell lysis component, the method only produces crude nucleic acid sample suitable for the specific PCR direct buffer of this patent, which contains certain amounts of nonionic surfactant, glycerol and Bovine Serum Albumin (BSA). These compounds are not contained in conventional commercial PCR buffers and may therefore interfere with the amplification of crude nucleic acid samples following sodium hydroxide lysis in conventional commercial PCR buffers. In view of this, there is an urgent need for new solvents having good cell degradation ability and DNA extraction ability while having no influence on the downstream PCR process.
The Deep Eutectic Solvent (DES) is a novel green solvent, has the advantages of cheap and rich raw material sources, simple synthesis, no need of purification of products and the like, is widely used for purifying proteins and nucleic acids by combining with other separation technologies, and is more environment-friendly and degradable than ionic liquid, so that more and more people pay attention to the novel solvent. A eutectic solvent can be prepared simply by mixing a Lewis acid and a base, or a hydrogen bond donor (hydrogen bond donor) and a hydrogen bond acceptor (hydrogen bond acceptor). Most importantly, the nature of the solvent can be adjusted as desired. The eutectic solvent is prepared to have a melting point lower than that of its constituent compounds and exist in a liquid state at room temperature, even though the constituent compounds are solid at room temperature. Imidazole is a five-membered aromatic heterocyclic compound, and the molecular structure of the imidazole contains two meta nitrogen atoms. Imidazole has acidity and also basicity, and in an environment with pH <6, the N atom at the 3 position is protonated, so that imidazole ring is positively charged, and when deprotonated under the condition with pH >6, uncharged imidazole ring is formed, and the binding capability of imidazole ring with DNA can be utilized as a delivery carrier of DNA.
Disclosure of Invention
The present invention is directed to a cell lysate directly used in PCR to solve the above problems of the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a cell lysate directly used in polymerase chain amplification reaction, comprising the steps of:
firstly, preparing a eutectic solvent, then adding a trace biological detection material into the eutectic solvent, incubating at the temperature of 50-60 ℃, shaking for 10-20 minutes, and directly adding the trace eutectic solvent into a PCR amplification system, wherein the amount 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 a still further scheme of the invention: the eutectic solvent consists of imidazole and alkylated carboxylic acid and water, the imidazole in the eutectic solvent exists in a positively charged state, the alkylated carboxylic acid has the property of dissolving cell membrane lipid, and the released DNA can be specifically combined with the imidazole. After the mixture is added to a PCR system, since the pH of a reaction buffer for PCR is generally 8 to 9, deprotonation of imidazole occurs in the system, causing the electrostatic interaction with DNA to disappear, DNA to be detached from imidazole, and PCR reaction to be performed.
As a still further scheme of the invention: molar ratio of imidazole to alkylated carboxylic acid 1: 2-2:1, stirring strongly at 60-80 ℃ under a closed condition until the solution becomes clear and transparent, adding water with the volume not more than 20% of the total volume into the system, and mixing uniformly.
As a still further scheme 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 volume of the PCR.
As a still further scheme of the invention: the molar ratio of imidazole to alkylated carboxylic acid is preferably from 1:2 to 1:1, and water is preferably added in an amount of 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 cracking cells by using the eutectic solvent 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 trace or trace lysate of biological samples, and is equivalent to or slightly superior to a magnetic bead reagent cracking and purifying method under certain conditions.
Drawings
FIG. 1 is a 500-fold diluted blood STR profile of imidazole/hexanoic acid eutectic solvent lysis;
FIG. 2 is a STR profile of the magnetic bead extraction reagent after extraction of 500-fold diluted blood.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to FIGS. 1-2, in one embodiment of the present invention, a cell lysate is directly used for PCR.
Example 1:
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 imidazole by using a magnetic stirrer at the temperature of 80 ℃ until clear and transparent liquid is formed, 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 by 500 times with physiological saline, dripping 1 microliter of the diluent on a glass slide, naturally drying, wiping the blood spot with a cotton swab or a flocked swab, locally shearing off the wiped cotton swab, putting the cotton swab into a 2.0 milliliter centrifuge tube, adding 100 microliter of imidazole/caproic acid eutectic solvent, completely immersing the sample, then heating, incubating and oscillating for 20 minutes at the temperature of 50 ℃, sucking 2 microliter of lysate, directly adding the lysate into an STR-PCR system for amplification, and the total amplification volume is 10 microliter. Simultaneously, 1 microliter of the diluted blood is used for DNA magnetic bead reagent cracking and purification, and 2 microliter of eluent is taken to be used for amplification in an STR-PCR system after the steps of cracking, adsorption, rinsing and elution, wherein the total amplification volume is 10 microliter.
Analysis of 50 samples of the two cracking methods respectively shows that 33 cases of more than 12 loci are detected in the STR typing map by using the magnetic bead reagent cracking and purifying method, all the 33 cases reach 16 loci, and the detection rate of effective individual identification is 68%. Over 12 loci are detected in 32 cases of 50 cases by using imidazole/caproic acid eutectic solvent to carry out STR typing detection, 16 loci are all detected, the detection rate for effective individual identification is 64 percent, and the peak values of sample maps are all in the range of 200-1000 RFU.
Example 2:
preparing a eutectic solvent: firstly, accurately weighing 13.6 g of imidazole, then adding the imidazole into a beaker filled with 28.8 g of caprylic acid, stirring the imidazole on a magnetic stirrer at 60 ℃ until clear and transparent liquid is formed, then adding 9.0 ml of deionized water into the system, and stirring the mixture for 10 minutes for later use.
Diluting human blood by 500 times with normal saline, dripping 1 microliter of the diluent on a glass slide, naturally drying, sticking the blood spot with a transparent adhesive tape, locally shearing the stuck adhesive tape, putting the partially cut adhesive tape into a 2.0 milliliter centrifuge tube, adding 100 microliter of imidazole/caprylic acid eutectic solvent, completely immersing the sample, then heating, incubating and oscillating for 10 minutes at the temperature of 60 ℃, sucking 3 microliter of lysate, directly adding the lysate into an STR-PCR system for amplification, and the total amplification volume is 10 microliter. Simultaneously taking 1 microliter of the diluted blood for DNA magnetic bead reagent cracking and purification, and taking 3 microliter of eluent for amplification in an STR-PCR system after the steps of cracking, adsorbing, rinsing and eluting, wherein the total amplification volume is 10 microliter.
Analysis of 50 samples of the two cracking methods respectively shows that 35 cases of more than 12 loci are detected in the STR typing map by using the magnetic bead reagent cracking and purifying method, all the 35 loci reach 16 loci, and the detection rate of effective individual identification is 80%. Over 12 loci and 38 loci detected by using imidazole/octanoic acid eutectic solvent for STR typing detection in 50 cases, wherein the loci reach 16 loci, the detection rate for effective individual identification is 76%, and the peak values of sample profiles are within the range of 400-1600 RFU.
Example 3:
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, and then 30 ml of deionized water is added into the system, and the mixture is stirred for 10 minutes for standby.
Diluting human blood by 500 times with normal saline, dripping 1 microliter of the diluent on a glass slide, naturally drying, sticking the blood spot with a transparent adhesive tape, locally shearing the stuck adhesive tape, putting the partially cut adhesive tape into a 2.0 milliliter centrifuge tube, adding 100 microliter of imidazole/butyric acid eutectic solvent, completely immersing the sample, then heating, incubating and oscillating for 15 minutes at the temperature of 55 ℃, sucking 1 microliter of lysate, directly adding the lysate into an STR-PCR system for amplification, and amplifying the total volume by 10 microliter. Simultaneously taking 1 microliter of the diluted blood for DNA magnetic bead reagent cracking and purification, and taking 1 microliter of eluent for amplification in an STR-PCR system after the steps of cracking, adsorbing, rinsing and eluting, wherein the total amplification volume is 10 microliter.
Analysis of 50 samples of the two cracking methods respectively shows that 32 cases of more than 12 loci are detected in the STR typing map by using the magnetic bead reagent cracking and purifying method, all the 32 cases reach 16 loci, and the detection rate of effective individual identification is 63%. Over 12 loci are detected in 32 cases of 50 cases of STR typing detection by using imidazole/butyric acid eutectic solvent, 16 loci are all detected, the detection rate for effective individual identification is 64 percent, and the peak values of sample maps are all in the range of 200-800 RFU.
It is noted that, herein, relational terms such as first and second, and the like may be 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. Also, 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 use of the phrase "comprising one of the elements does not exclude the presence of other like elements in the process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A cell lysate directly used in polymerase chain amplification reaction, comprising the steps of:
firstly, preparing a eutectic solvent, then adding a trace biological detection material into the eutectic solvent, incubating at the temperature of 50-60 ℃, shaking for 10-20 minutes, and directly adding the trace eutectic solvent into a PCR amplification system, wherein the amount 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.
2. The cell lysate directly used in PCR amplification reaction according to claim 1, wherein the eutectic solvent consists of imidazole and alkylated carboxylic acid, the imidazole in the eutectic solvent exists in a positively charged state, and the alkylated carboxylic acid has the property of dissolving cell membrane lipids, the released DNA can be specifically bound to the imidazole, and after the mixture is added into the PCR system, due to the pH of the reaction buffer solution of PCR being usually 8-9, the imidazole is deprotonated in the system, so that the electrostatic interaction with DNA is disappeared, DNA is separated from imidazole, and PCR reaction is performed.
3. The cell lysate directly used in the pcr amplification reaction of claim 1, wherein the eutectic solvent is prepared according to the method and composition, and the molar ratio of imidazole to alkylated carboxylic acid is 1: 2-2:1, stirring strongly at 60-80 ℃ under a closed condition until the solution becomes clear and transparent, adding water with the volume not more than 20% of the total volume into the system, and mixing uniformly.
4. The cell lysate directly used in PCR as claimed in claim 1, wherein the mass ratio of the micro biological sample to the eutectic solvent is preferably 1:90, and the volume of the eutectic solvent added to the PCR system is preferably 20% of the volume of the PCR.
5. A cell lysate directly used in PCR amplification reaction according to claim 3, wherein the molar ratio of imidazole to alkylated carboxylic acid is preferably 1:2 to 1:1, and the amount of water added is preferably 10%. The eutectic solvent has low melting point and low viscosity.
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| US20150299769A1 (en) * | 2012-11-07 | 2015-10-22 | Qiagen Gmbh | Method for lysing a fixed biological sample |
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