CN106987588B - Virus/bacteria lysate and fluorescent quantitative PCR detection method - Google Patents
Virus/bacteria lysate and fluorescent quantitative PCR detection method Download PDFInfo
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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
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a virus/bacteria lysate and a fluorescent quantitative PCR detection method, belonging to the field of biological detection. The lysis solution is prepared by uniformly mixing NaCl, triton X-100, lithium dodecyl sulfate, EDTA-2Na and purified water. The lysate of the invention can omit the step of DNA purification in the PCR reaction process, the nucleic acid extraction and amplification detection of the sample are carried out in a reaction tube, the separation or purification operations such as heating, centrifugation, oscillation and the like are not needed, the operation process is simple, convenient and low in cost, the requirement on operators is extremely low, the detection time is greatly shortened, and the lysate is particularly suitable for bedside diagnosis of major diseases and field inspection and quarantine work when major epidemic outbreaks occur.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction and detection of viruses and bacteria, in particular to lysate for extracting nucleic acid of viruses and bacteria and a detection method for performing fluorescence quantitative PCR by using the lysate.
Background
In the prior art, the methods for extracting viruses from serum and plasma mainly comprise the following steps:
firstly, boiling method: adding lysis solution into virus and bacteria samples such as serum or plasma, boiling in water bath or dry bath, centrifuging at high speed, and taking supernatant as PCR template, with the advantages of simple operation, high content of extracted nucleic acid impurities, and easy inhibition of later-stage PCR amplification, and high local pressure generated by boiling easily causes aerosol pollution, resulting in undivided detection result.
II, centrifugal column extraction: adding high-salt low-pH lysis solution into virus and bacteria samples such as serum or plasma, adsorbing by using a nucleic acid adsorption membrane, washing for 2-3 times by using a washing solution, and finally eluting nucleic acid by using low-salt high-pH eluent to serve as a PCR template. The method has high purity of extracted nucleic acid, but has the defects of multiple times of high-speed centrifugation, complicated manual operation, low efficiency and incapability of realizing automation.
And thirdly, magnetic bead method: the magnetic bead method can be regarded as the improvement of the centrifugal column method, wrap the absorbing membrane with the functional substance on the magnetic nanoparticle, virus, bacterium sample such as serum or plasma, etc. through adding the lysis solution of the low pH value of the high salt, then use the nanometer magnetic particle coated with the nucleic acid absorbing membrane to absorb, use the washing liquid to wash for 2-3 times, use the eluant of the high pH value of the low salt to elute the nucleic acid as PCR template finally. The method has high purity of extracted nucleic acid and can realize automatic extraction, but the technology needs higher experimental skills of operators and is not easy to popularize, and if the method is provided with automatic equipment, the detection cost is increased.
In the extraction methods, a chemical method is adopted to realize cell disruption and nucleic acid release, and the nucleic acid is separated and purified based on the characteristic that the nucleic acid is more hydrophilic than other biomacromolecules such as protein, polysaccharide, fat and the like, so that the purification step has long operation time and high cost.
Disclosure of Invention
The invention aims to provide a virus/bacteria lysate and a fluorescence quantitative PCR detection method which are simple to operate and low in cost, can meet the requirement that most of common viruses and bacteria in clinic can be directly subjected to fluorescence quantitative PCR detection without nucleic acid purification, and is used for solving the problems of long purification time, complex operation and high cost in the existing nucleic acid extraction process.
In order to realize the purpose, the method adopts a lysis solution which can rapidly lyse virus and bacterial coat protein to release genetic materials of the virus and bacterial coat protein, and directly adds the lysis solution into corresponding PCR reaction solution, thereby realizing quantitative or qualitative detection aiming at pathogenic microorganisms to be detected. Specifically, the virus/bacterium lysis solution is prepared by uniformly mixing NaCl, triton X-100, lithium dodecyl sulfate, EDTA-2Na and purified water, wherein the final concentration of each component is as follows: NaCl is 0.8-1.2M, triton X-100 is 10-15 v%, lithium dodecyl sulfate is 0.01-0.05% w/v (mass-volume ratio), and EDTA-2Na is 5-10 mM.
Preferably, the final concentration ratio of each component is as follows: NaCl 1M, triton X-100 10 v.%, lithium lauryl sulfate 0.02%, EDTA-2Na 5 mM.
Preferably, the final concentration ratio of each component is as follows: NaCl 1M, triton X-100 12 v.%, lithium lauryl sulfate 0.04%, EDTA-2Na 8 mM.
In the technical scheme, the high-concentration sodium chloride is adopted to be beneficial to salting out and denaturation of protein so as to separate the protein from nucleic acid; triton X-100 and lithium dodecyl sulfate belong to nonionic and ionic surfactants respectively, which are beneficial to splitting virus coat protein and separating nucleic acid from protein, wherein the nonionic surfactant does not generate obvious inhibition on later PCR, so the volume concentration is preferably 10-15 v.%, more preferably 10 v.%, and the ionic surfactant can inhibit PCR at high dose, so 0.01-0.05% w/v (mass-to-volume ratio) is preferably used, and 0.04% w/v is more preferably used. EDTA-2Na can form a chelate with the divalent metal ion in the sample, thereby leaving the DNase free of catalyst and indirectly protecting the DNA from degradation. The components have synergistic effect, and the virus or bacteria can be directly amplified by PCR after being cracked, thereby bypassing the process of nucleic acid purification.
The invention also provides a virus/bacterium fluorescence quantitative PCR detection method, which comprises the following steps:
A. preparing a lysate according to the ratio of the virus/bacteria lysate;
B. extraction of viral/bacterial nucleic acids: adding the lysate obtained in the step A and a serum or plasma sample to be detected into a PCR reaction tube, uniformly mixing, and standing for 5-15 min to obtain a DNA sample of virus/bacteria;
C. and (3) PCR amplification: and D, adding the PCR reaction solution into the PCR reaction tube of the DNA sample obtained in the step B, placing the PCR reaction tube on a fluorescent quantitative PCR instrument, setting circulation parameters, and carrying out PCR amplification and fluorescent detection.
The method for preparing the lysate in the step A comprises the following steps:
adding 46.8-70.2 g (namely 0.8-1.2 mol) of sodium chloride, 100-150 mL of triton X-100, 1-5 g of lithium dodecyl sulfate and 10-20 mL of 0.5M EDTA-2Na with the pH value of 8.0 into a container, then adding 600mL of purified water, uniformly mixing, and fixing the volume to 1000 mL.
The PCR reaction solution in the step B comprises: PCR Buffer solution (PCR Buffer), MgCl2dNTPs, a primer pair and a corresponding probe for detecting virus/bacteria, and Taq DNA polymerase or enzyme mixed solution.
The method of the invention has the following advantages: the lysate of the invention can omit the step of DNA purification in the PCR reaction process, the nucleic acid extraction and amplification detection of the sample are carried out in a reaction tube, the separation or purification operations such as heating, centrifugation, oscillation and the like are not needed, the operation process is simple, convenient and low in cost, the requirement on operators is extremely low, the detection time is greatly shortened, and the lysate is particularly suitable for bedside diagnosis of major diseases and field inspection and quarantine work when major epidemic outbreaks occur.
Drawings
FIG. 1 is an amplification curve of 4-gradient E.coli in example 2, in which the abscissa represents the cycle number and the ordinate represents the fluorescence intensity.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
The application of the lysate of the present invention is illustrated by taking the fluorescent quantitative PCR detection of porcine circovirus type 2 (PCV-2) as an example.
A. Preparing a lysate: 58.5 g of sodium chloride, 100mL of triton X-100 (polyethylene glycol octylphenyl ether), 2 g of lithium dodecyl sulfate, 10mL of an EDTA-2Na solution with a concentration of 0.5M, pH and a value of 8.0, 600mL of purified water were added to the vessel, and after mixing, the vessel was transferred to a volumetric flask to reach a volume of 1000 mL.
B. Extraction of viral/bacterial nucleic acids: adding 5 mu L of lysate prepared in the step A and 5 mu L of serum or plasma sample to be detected into a PCR reaction tube, repeatedly beating the lysate and the plasma sample for 3 times by using a pipettor, uniformly mixing the lysate and the plasma sample, and standing the mixture for 10min to obtain a DNA sample of bacteria;
C. and (3) PCR amplification: and D, adding 40 mu L of prepared PCR reaction liquid into the PCR reaction tube of the DNA sample obtained in the step B, placing the PCR reaction tube on a fluorescent quantitative PCR instrument, setting circulation parameters, and carrying out PCR amplification and fluorescent detection, wherein the PCR amplification reaction program is shown in Table 1. In this example, a fluorescent quantitative PCR instrument of type SLAN-48P, a macrolite, was used for detection.
Meanwhile, purified water is used as a negative control, a culture solution containing porcine circovirus type 2 bacteria is used as a positive control, and PCR amplification and fluorescence detection are respectively carried out according to the conditions.
TABLE 1PCR reaction procedure and conditions
The components and concentrations of the PCR reaction solution are shown in Table 2.
TABLE 2 composition and concentration of PCR reaction solution
| Components | Single dose (mu L) |
| 5×PCR Buffer | 10 |
| MgCl2(1M) | 0.5 |
| dNTPs(25mM) | 0.5 |
| PCV-2-F(50pmol/μL) | 0.4 |
| PCV-2-R(50pmol/μL) | 0.4 |
| PCV-2-P(50pmol/μL) | 0.2 |
| Taq DNA polymerase (5U/. mu.L) | 1 |
| Purified water | Is complemented to 40 |
The PCR Buffer comprises the following components in concentration: 250mM tris HCl, pH 8.0,375mM KCl, 25% (by volume) glycerol, 25% ammonium sulfate (by volume).
The upstream primer PCV-2-F and the downstream primer PCV-2-R for amplifying the porcine circovirus type 2 and the probe PCV-2-P for detecting the amplified porcine circovirus type 2 can adopt mature nucleotide sequences in the prior art, and the nucleotide sequences adopted in the embodiment are shown in Table 3.
TABLE 3 primer and Probe sequences for the detection of porcine circovirus type 2
54 porcine circovirus type 2 serum samples were tested as described above. Meanwhile, as a control, the nucleic acid purification is carried out on each sample by a centrifugal column extraction method, which comprises the following steps: adding a high-salt low-pH lysis solution into a serum sample, adsorbing by using a nucleic acid adsorption membrane, washing for 2-3 times by using a washing solution, eluting nucleic acid by using a low-salt high-pH eluent as a PCR template, adding the PCR reaction solution prepared in the table 2, and performing amplification and detection according to the PCR reaction procedures and conditions in the table 1. In this example, the FinePure virus DNA/RNA column extraction kit from Jivan Biotechnology (Beijing) Ltd is used.
The results of the tests of this example and the control example were compared, see table 4.
Table 4 comparison of test results of example 1 and comparative example
The above results show that: the cracking agent and the corresponding method of the embodiment are close to the detection positive rate of the classical spin column extraction, and the embodiment is simple to operate, and can avoid the occurrence of false negative results caused by errors generated in the spin column extraction process.
Example 2
A. Preparing a lysate: 58.5 g of sodium chloride, 150mL of triton X-100, 4 g of lithium dodecyl sulfate, 16mL of EDTA-2Na solution with the concentration of 0.5M, pH being 8.0, 600mL of purified water were added to the container, and after mixing, the mixture was transferred to a volumetric flask to reach a volume of 1000 mL.
B. Preparing a sample to be tested: diluting Escherichia coli liquid with known concentration to 1 × 107cfu/ml、1×106cfu/ml、1×105cfu/ml、1×104cfu/mL four 10-fold dilution gradient concentrations.
Taking a plurality of PCR reaction tubes, respectively adding 5 mu L of lysis solution prepared in the step A, sequentially adding 5 mu L of escherichia coli liquid with the four concentrations, repeatedly beating by using a pipettor for 3 times, uniformly mixing, and standing for 10min to obtain a virus lysis system;
C. and (3) PCR amplification: and C, adding 40 mu L of prepared PCR reaction solution into the virus lysis system in the step B, amplifying according to the PCR reaction program and conditions in the table 1, and detecting.
Purified water was used as a negative control.
The composition and concentration of the PCR reaction solution of this example are shown in Table 5.
TABLE 5 composition and concentration of PCR reaction solution
| Components | Single dose (mu L) |
| 5×PCR Buffer | 10 |
| MgCl2(1M) | 0.5 |
| dNTPs(25mM) | 0.5 |
| Ec-F(50pmol/μL) | 0.2 |
| Ec-R(50pmol/μL) | 0.2 |
| Ec-P(50pmol/μL) | 0.1 |
| Taq DNA polymerase (5U/. mu.L) | 1 |
| Rox(50μM) | 0.1 |
| Purified water | Is complemented to 40 |
The concentration and composition of the 5 XPCR Buffer in this example are the same as in example 1.
The technology for detecting the upstream primer Ec-F, the downstream primer Ec-R and the probe Ec-P of the escherichia coli is mature in the prior art. The nucleotide sequences used in this example are shown in Table 6.
TABLE 6 primer and Probe sequences for amplification of circovirus type 2
The amplification results are shown in FIG. 1, and the Ct values obtained are shown in Table 7.
TABLE 7 results of detection of Escherichia coli
| Escherichia coli solution | Test results (Ct value) |
| 1×107cfu/ml | 20.01 |
| 1×106cfu/ml | 23.11 |
| 1×105cfu/ml | 26.73 |
| 1×104cfu/ml | 29.88 |
The above results show that: the lysate of this example has good sensitivity to bacterial samples and the Ct value changes in a gradient with decreasing concentration.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Jivan Biotechnology (Beijing) Ltd
<120> lysis solution of virus/bacteria and fluorescent quantitative PCR detection method
<130>2010
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Claims (7)
1. The virus/bacterium lysis solution is characterized in that the lysis solution is prepared by uniformly mixing NaCl, triton X-100, lithium dodecyl sulfate, EDTA-2Na and purified water, and the final concentration of each component is as follows: NaCl is 1-1.2M, triton X-100 is 10-12 v%, lithium dodecyl sulfate is 0.01-0.04% w/v, and EDTA-2Na is 5-8 mM.
2. The virus/bacterium lysate according to claim 1, wherein the final concentration ratio of each component is: NaCl 1M, triton X-100 10 v.%, lithium lauryl sulfate 0.02%, EDTA-2Na 5 mM.
3. The virus/bacterium lysate according to claim 1, wherein the final concentration ratio of each component is: NaCl 1M, triton X-100 12 v.%, lithium lauryl sulfate 0.04%, EDTA-2Na 8 mM.
4. A fluorescent quantitative PCR detection method for virus/bacteria non-disease diagnosis is characterized by comprising the following steps:
A. preparing a lysate according to the virus/bacterium lysate ratio of claim 1;
B. extraction of viral/bacterial nucleic acids: adding the lysate obtained in the step A and a serum or plasma sample to be detected into a PCR reaction tube, uniformly mixing, and standing for 5-15 min to obtain a DNA sample of virus/bacteria;
C. and (3) PCR amplification: and D, adding the PCR reaction solution into the PCR reaction tube of the DNA sample obtained in the step B, placing the PCR reaction tube on a fluorescent quantitative PCR instrument, setting circulation parameters, and carrying out PCR amplification and fluorescent detection.
5. The method for fluorescent quantitative PCR detection of virus/bacteria non-disease diagnosis according to claim 4, wherein the method for preparing the lysate in step A is as follows:
adding 58.5-70.2 g of sodium chloride, 100-120 mL of triton X-100, 1-4 g of lithium dodecyl sulfate and 10-16 mL of 0.5M EDTA-2Na with the pH value of 8.0 into a container, then adding 600mL of purified water, uniformly mixing, and fixing the volume to 1000 mL.
6. The fluorescence quantitative PCR detection method for non-disease diagnosis of virus/bacteria according to claim 4, wherein 5 μ L of each of the lysate and the serum or plasma sample to be detected is added to the PCR reaction tube in step B, mixed well, and left to stand at room temperature for 10 min.
7. The method for fluorescent quantitative PCR detection of non-disease diagnosis of virus/bacteria according to claim 4, wherein the PCR reaction solution in step C comprises: PCR buffer solution, MgCl2, dNTPs, primer pairs and corresponding probes for detecting virus/bacteria, and Taq DNA polymerase or enzyme mixture.
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| CN114426966A (en) * | 2020-10-29 | 2022-05-03 | 西南大学 | Cell lysate, kit and method for rapidly and non-toxically extracting genomic DNA of animals, plants or microorganisms |
| CN113684251A (en) * | 2021-08-27 | 2021-11-23 | 张乃元 | New application of PT-1 or effective components thereof in pathogenic microorganism detection and PT-1-based nucleic acid detection lysate |
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| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN105420230A (en) * | 2016-01-14 | 2016-03-23 | 北京纳捷诊断试剂有限公司 | Lysis solution for extracting nucleic acid through magnetic bead method |
| CN105420403A (en) * | 2016-01-14 | 2016-03-23 | 北京纳捷诊断试剂有限公司 | Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube |
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| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN105420230A (en) * | 2016-01-14 | 2016-03-23 | 北京纳捷诊断试剂有限公司 | Lysis solution for extracting nucleic acid through magnetic bead method |
| CN105420403A (en) * | 2016-01-14 | 2016-03-23 | 北京纳捷诊断试剂有限公司 | Real-time fluorescence quantification PCR method with magnetic bead nucleic acid extraction and amplification conducted in one tube |
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