CN113930418A - Nucleic acid releasing agent and method for releasing nucleic acid - Google Patents
Nucleic acid releasing agent and method for releasing nucleic acid Download PDFInfo
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- CN113930418A CN113930418A CN202111196322.4A CN202111196322A CN113930418A CN 113930418 A CN113930418 A CN 113930418A CN 202111196322 A CN202111196322 A CN 202111196322A CN 113930418 A CN113930418 A CN 113930418A
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- C—CHEMISTRY; METALLURGY
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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
Abstract
The invention relates to a nucleic acid releasing agent and a nucleic acid releasing method thereof, the scheme comprises 0.5-1.5M Tris-HCl, 30-70mM EDTA, 0.5-1.5M cysteine, 10-30U/. mu.L RRI, DEPC water, NP-40 with the volume percentage concentration of 15-30% and Poly A with the volume percentage concentration of 3-7%, wherein the RRI is an RNase inhibitor. Balancing the nucleic acid releasing agent to room temperature and shaking up; adding the nucleic acid releasing agent after shaking up into a PCR reaction tube; and adding a sample to be detected, blowing, beating and uniformly mixing to obtain the target nucleic acid, wherein the volume ratio of the nucleic acid releasing agent to the sample to be detected is 1: 3. The method has the advantages of high recovery rate, simplicity in operation and low production cost.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a nucleic acid releasing agent and a nucleic acid releasing method thereof.
Background
PCR (Polymerase Chain Reaction) is a molecular biology technology for amplifying specific nucleic acid fragments, and is mainly characterized in that a large amount of trace nucleic acid can be enriched and increased, so that the purpose of conveniently detecting the trace nucleic acid is achieved. The PCR method commonly used in medical diagnosis is mainly a real-time fluorescent quantitative PCR (qPCR) method based on a dual-fluorescence probe, and targets for in vitro diagnosis by using the qPCR method mainly comprise human genome DNA, DNA viruses, bacteria, fungi, RNA viruses and the like. Because the structure of RNA is a single-stranded structure, which is unstable and easy to degrade, the requirement in the sample treatment process is very high, and a complex method is needed to perform pretreatment, nucleic acid extraction and purification on the RNA sample to be amplified, so that after pure nucleic acid is obtained, detection can be performed to obtain a stable result.
Therefore, the current nucleic acid extraction methods applied to fluorescent quantitative PCR amplification mainly comprise the following 4 methods:
(1) the traditional boiling method comprises the following steps: uniformly mixing the lysate and the blood sample, performing pyrolysis, and centrifuging to obtain nucleic acid;
(2) concentration and boiling method: firstly, polysaccharide is used for concentrating and precipitating virus nucleic acid, then lysate is added for boiling, and the nucleic acid is obtained through centrifugation;
(3) centrifugal column method: adsorbing nucleic acid by using a chromatographic column after cracking, and eluting to obtain pure nucleic acid;
(4) magnetic bead method: adsorbing nucleic acid with magnetic beads, and eluting to obtain relatively pure nucleic acid.
The first 2 extraction methods have the disadvantages of requiring multiple tube transfer, pipetting, centrifugation, etc., consuming long time for sample processing, and inevitably losing part of nucleic acid in the operation of high temperature processing, resulting in a low value of the transportation amount of the nucleic acid sample finally used for PCR amplification. Although the purity of nucleic acid extracted by the centrifugal column method is greatly improved as compared with the above two boiling methods, many manual steps still have a problem of low efficiency. The magnetic bead method has the advantages of simple extraction steps, high recovery rate, good purity, easy automation, high product price and low application range at present.
Therefore, a nucleic acid releasing agent and a method for releasing nucleic acid thereof, which have high recovery efficiency, simple operation and low production cost, are desired.
Disclosure of Invention
The present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a nucleic acid releasing agent and a nucleic acid releasing method thereof.
In order to realize the purpose of the invention, the invention adopts the following technical scheme: the nucleic acid releasing agent comprises 0.5-1.5M Tris-HCl, 30-70mM EDTA, 0.5-1.5M cysteine, 10-30U/. mu.L RRI, DEPC water, NP-40 with the volume percentage concentration of 15-30% and Poly A with the volume percentage concentration of 3-7%, wherein the RRI is an RNase inhibitor.
The working principle and the beneficial effects are as follows: 1. compared with the prior art, the nucleic acid releasing agent of this application is totally different with traditional nucleic acid releasing agent composition, and this application regards polyA as the nucleic acid settling agent, and cysteine and NP-40 are as nonionic activator to cooperate with polyA to release the nucleic acid of the sample that awaits measuring, specifically, cysteine and NP-40 are in this scheme: the two are used as nonionic surfactants to crack cell membranes so as to quickly release nucleic acid, and in addition, NP-40 can play a role in protecting reverse transcriptase so that the reverse transcriptase can normally work in an alkaline environment, so that a lysate is not required to be boiled in use, a centrifugal column method and a magnetic bead method are not required to be adopted for extracting nucleic acid, and the nucleic acid can be obtained only by mixing the nucleic acid and a sample to be detected in a PCR reaction tube, so that the operation difficulty can be obviously reduced, the operation time can be reduced, the waste of the nucleic acid can be reduced, and the advantages of high extraction efficiency, simplicity in operation, capability of completing sample treatment within 3 minutes at room temperature and low production cost can be achieved;
2. the scheme provides the nucleic acid releasing agent which can directly release the nucleic acid for PCR detection, and the nucleic acid releasing agent can be directly used for PCR detection, more nucleic acid extracting agents related to a nucleic acid magnetic bead extraction method exist in the prior art, however, the nucleic acid magnetic bead extraction method needs to be carried out by matching with a nucleic acid extracting instrument and a magnetic frame, the general operation time of a program is about 30 minutes, the cost is high, the time is long, and the scheme provides the nucleic acid releasing agent which can directly release the nucleic acid for PCR detection.
3. And the preparation method of the nucleic acid releasing agent is very simple, and the nucleic acid releasing agent can be prepared by mixing and shaking the above various costs, so that the production cost and the preparation difficulty are obviously reduced.
Further, it was composed of 1M Tris-HCl, 50mM EDTA, 1M cysteine, 20U/. mu.L RRI, DEPC water, NP-40 at 25% by volume, and Poly A at 5% by volume.
The nucleic acid releasing method adopting the nucleic acid releasing agent specifically comprises the following steps:
balancing the nucleic acid releasing agent to room temperature and shaking up;
adding the nucleic acid releasing agent after shaking up into a PCR reaction tube;
and adding a sample to be detected, blowing, beating and uniformly mixing to obtain the target nucleic acid, wherein the volume ratio of the nucleic acid releasing agent to the sample to be detected is 1: 3. Compared with the prior art, the method has the advantages that the target nucleic acid can be obtained only by shaking the nucleic acid releasing agent and the sample to be detected uniformly in the PCR reaction tube according to the volume ratio of 1:3 without independently extracting the nucleic acid of the sample, so that the operations of centrifuging, discarding supernatant, rotating the tube and the like in the prior art are completely avoided, the experimental steps are greatly simplified, and the operations of boiling, transferring the reaction container and the like do not exist, so that the nucleic acid loss does not exist, the recovery rate of the nucleic acid is greatly improved, and the detection sensitivity is also greatly improved.
Further, the sample to be tested includes whole blood, serum, plasma or secretion.
By the arrangement, the nucleic acid releasing agent can be used for quickly and accurately measuring the content of nucleic acid in unknown samples such as whole blood, serum, plasma or secretion, and has the advantages of good repeatability, strong specificity and good compatibility.
Further, the target nucleic acid is DNA or RNA.
Further, the nucleic acid releasing agent is prepared by the following method:
1 μ L of 1M Tris-HCl, 0.4 μ L of 50mM EDTA, 0.2 μ L of 1M cysteine, 0.2 μ L of 25% NP-40, 0.2 μ L of 5% Poly A, 0.04 μ L of 20U/. mu.L RRI, and 7.96 μ L DEPC water were mixed well.
The setting proves that the dosage of the nucleic acid releasing agent is very small, the advantages of low cost and safe use can be kept on the premise of easy discrimination, the fluorescent quantitative PCR instrument can be suitable for different types of fluorescent quantitative PCR instruments, particularly POCT, and can be widely applied to the fields of detection of pathogenic microorganisms, gene mutation detection, pet disease detection, forensic identification and the like.
Drawings
FIG. 1 is a schematic view showing detection of ORF1ab gene by the nucleic acid releasing agent and magnetic bead method in example 1 of the present invention;
FIG. 2 is a schematic diagram of detection of ORF1ab gene by the nucleic acid releasing agent of example 2 and the nucleic acid releasing agent of example 1;
FIG. 3 is a schematic diagram of detection of ORF1ab gene by the nucleic acid releasing agent of example 3 and the nucleic acid releasing agent of example 1;
FIG. 4 is a schematic diagram of detection of ORF1ab gene by the nucleic acid releasing agent of example 4 and the nucleic acid releasing agent of example 1;
FIG. 5 is a schematic view of detection of N gene by the nucleic acid releasing agent and magnetic bead method in example 1;
FIG. 6 is a schematic diagram of detection of N gene by the nucleic acid releasing agent of example 2 and the nucleic acid releasing agent of example 1;
FIG. 7 is a schematic view of detection of N gene by the nucleic acid releasing agent of example 3 and the nucleic acid releasing agent of example 1;
FIG. 8 is a schematic view of detection of N gene by the nucleic acid releasing agent of example 4 and the nucleic acid releasing agent of example 1;
FIG. 9 is a schematic diagram of the detection of ORF1ab gene by the magnetic bead method (rapid PCR);
FIG. 10 is a schematic diagram of detection of ORF1ab gene (rapid PCR) in example 1;
FIG. 11 is a schematic diagram of detection of ORF1ab gene (rapid PCR) in example 2;
FIG. 12 is a schematic diagram of detection of ORF1ab gene (rapid PCR) in example 3;
FIG. 13 is a schematic diagram of detection of ORF1ab gene (rapid PCR) in example 4;
FIG. 14 is a schematic diagram of detection of N gene by the magnetic bead method (rapid PCR);
FIG. 15 is a schematic diagram of detection of N gene (rapid PCR) in example 1;
FIG. 16 is a schematic diagram of detection of N gene (rapid PCR) in example 2;
FIG. 17 is a schematic diagram of detection of N gene (rapid PCR) in example 3;
FIG. 18 is a schematic diagram of detection of N gene (rapid PCR) in example 4.
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 that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Example 1: the specific embodiment provided by the scheme is as follows:
the nucleic acid releasing agent consists of 1M Tris-HCl, 50mM EDTA, 1M cysteine, 20U/. mu.L RRI, DEPC water, NP-40 with the volume percentage concentration of 25 percent and Poly A with the volume percentage concentration of 5 percent.
For example, the preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
1 μ L of 1M Tris-HCl, 0.4 μ L of 50mM EDTA, 0.2 μ L of 1M cysteine, 0.2 μ L of 25% NP-40, 0.2 μ L of 5% Poly A, 0.04 μ L of 20U/. mu.L RRI, and 7.96 μ L DEPC water were mixed well.
Wherein, 1M Tris-HCl is 1mol/L Tris hydroxymethyl aminomethane hydrochloride; the 50mM EDTA is 50mmol/L EDTA; RRIs are rnase inhibitors; DEPC water is ultrapure water (primary water) treated with DEPC (diethyl pyrocarbonate) and sterilized at high temperature and high pressure, and is colorless liquid and free of impurity RNA, DNA and protein; NP-40 is an abbreviation for Nonidet P40, which is translated in Chinese to ethylphenylpolyethyleneglycol, a mild nonionic detergent; poly A acts as a nucleic acid precipitating agent.
Example 2: blank set of experiments lacking cysteine and NP-40
The nucleic acid releasing agent consists of 1M Tris-HCl, 50mM EDTA, 20U/. mu.L RRI, DEPC water and 5% Poly A by volume percentage concentration.
For example, the preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
the mixture was prepared by mixing 1. mu.L of 1M Tris-HCl, 0.4. mu.L of 50mM EDTA, 0.2. mu.L of 5 vol.% Poly A, 0.04. mu.L of 20U/. mu.L RRI, and 8.36. mu.L DEPC water, respectively.
Example 3: control experiment with cysteine substituted for sodium lauroyl sarcosinate
The nucleic acid releasing agent consists of 1M Tris-HCl, 50mM EDTA, 1M sodium lauroyl sarcosine, 20U/. mu.LRRI, DEPC water, NP-40 with the volume percentage concentration of 25 percent and Poly A with the volume percentage concentration of 5 percent.
For example, the preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
the mixture was prepared by thoroughly mixing 1. mu.L of 1M Tris-HCl, 0.4. mu.L of 50mM EDTA, 0.2. mu.L of 1M sodium lauroyl sarcosine, 0.2. mu.L of 25% NP-40, 0.2. mu.L of 5% Poly A, 0.04. mu.L of 20U/. mu.L RRI, and 7.96. mu.L of DEPC water, respectively.
Example 4: control experiments with NP-40 replaced with Triton X-100:
the nucleic acid releasing agent consists of 1M Tris-HCl, 50mM EDTA, 1M cysteine, 20U/. mu.L RRI, DEPC water, 25% Triton X-100 by volume percentage and 5% Poly A by volume percentage.
For example, the preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
the mixture was prepared by mixing 1. mu.L of 1M Tris-HCl, 0.4. mu.L of 50mM EDTA, 0.2. mu.L of 1M cysteine, 0.2. mu.L of 25% Triton X-100, 0.2. mu.L of 5% Poly A, 0.04. mu.L of 20U/. mu.L RRI, and 7.96. mu.L DEPC water, respectively.
It is worth noting that the configuration environments and conditions of the above embodiments 1 to 4 are completely consistent.
Example 5
The nucleic acid releasing method using the nucleic acid releasing agent of the above example 1 to example 4 specifically includes the following steps:
step 1, balancing a nucleic acid releasing agent to room temperature and then shaking up;
and 3, adding a sample to be detected, blowing, beating and uniformly mixing to obtain target nucleic acid, wherein the volume ratio of the nucleic acid releasing agent to the sample to be detected is 1:3, the sample to be detected comprises whole blood, serum, plasma or secretion, and the target nucleic acid is DNA or RNA.
Compared with the prior art, the nucleic acid releasing agent is completely different from the traditional nucleic acid releasing agent in components, the PolyA is used as a nucleic acid precipitation aid in the application, the cysteine and the NP-40 are used as nonionic active agents to be matched with the PolyA to release the nucleic acid of a sample to be detected, the nucleic acid releasing agent is similar to the nucleic acid extraction by a magnetic bead method, but the operation methods are completely different, so that a lysis solution is not required to be used for boiling in use, the nucleic acid is not required to be extracted by a centrifugal column method and a magnetic bead method, and the nucleic acid can be obtained only by mixing the nucleic acid and the sample to be detected in a PCR reaction tube, so that the operation difficulty can be obviously reduced, the operation time can be reduced, the nucleic acid waste can be reduced, and the advantages of high extraction efficiency, simplicity in operation and low production cost can be achieved.
Example 6
In this example, the nucleic acid releasing agent and the nucleic acid releasing method of the present application were verified by comparing the nucleic acid releasing agent of examples 1 to 4 with the nucleic acid extracted by the conventional magnetic bead method and by using the general PCR (LC480) program.
The instrument used for the experiment is an LC480 real-time fluorescence quantitative PCR instrument which is purchased from Roche diagnostics GmbH; an iRapid4 real-time fluorescence quantitative PCR instrument is manufactured by Hangzhou Dian biotechnology limited; the nucleic acid extraction instrument EB1000 was manufactured by Hangzhou Dian Biotechnology Ltd;
the nucleic acid releasing agent and the nucleic acid extracting or purifying reagent (magnetic bead method) are both produced by Hangzhou dean biotechnology, Inc. (I am), the reagent purity is molecular biology grade, the reagent and enzyme system used by the PCR reaction solution are purchased from Zhuhaibaorui biotechnology, Inc., and the primer and the probe are both synthesized by Shanghai Baili George biotechnology, Inc.; the new coronavirus is customized and synthesized by Baibai Australia (Suzhou) Biotechnology limited and provides a quality inspection report.
Detecting 2019-nCoV nucleic acid quality control product A (1E 7-1E 4 copies/mL) by using a nucleic acid releasing agent and extracting nucleic acid by using a magnetic bead method to detect 2019-nCoV nucleic acid quality control product B (1E 7-1E 4 copies/mL).
1. Reagent preparation
(1) Example 1-nucleic acid releasing agent formulation of example 4:
the components of the nucleic acid releasing agent provided in example 1 are as follows:
table 1 nucleic acid releasing agent provided in example 1
| Name of reagent | Volume (per person) |
| 1M Tris-HCl | 1μL |
| 50mM EDTA | 0.4μL |
| 1M cysteine | 0.2μL |
| 25％NP-40 | 0.2μL |
| 5％Poly A | 0.2μL |
| 20U/μL RRI | 0.04μL |
| DEPC water | 7.96μL |
The preparation method of the nucleic acid releasing agent comprises the following steps: the components are measured according to the above table 1 and added in sequence, and the mixture is fully and evenly mixed to prepare the composition.
The formulation of the nucleic acid releasing agent of examples 2 to 4 is the same as that described in the above examples and will not be redundantly described here.
(2) Preparation of nucleic acid extraction or purification reagents (magnetic bead method):
the nucleic acid extraction or purification reagent produced by Hangzhou dean biotechnology limited is mainly composed of lysis solution, washing solution I, washing solution II, washing solution III, magnetic bead suspension, eluent, protease K and a magnetic rod sleeve. Nucleic acid extraction was performed in the manner indicated in the specification.
(3) Preparing a PCR reaction solution:
the addition amounts of the components of the PCR reaction solution are shown in the following table 2:
table 2: PCR reaction solution Components
Wherein Fast Direct RT Premix Buffer II (dUTP) (Mg2+ free) (DG) contains (dATP, dTTP, dCTP, dGTP and dUTP), the template is taken to be 3 muL, and the reaction system is 25 muL.
Wherein, the primer probe sequence is as follows:
ORF1ab-F：CCCTGTGGGTTTTACACTTAA
ORF1ab-R：ACGATTGTGCATCAGCTGA
ORF1ab-P：FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1
N-F：GGGGAACTTCTCCTGCTAGAAT
N-R：CAGACATTTTGCTCTCAAGCTG
N-P：VIC-TTGCTGCTGCTTGACAGATT–BHQ1
RNP-F：AGATTTGGACCTGCGAGCG
RNP-F：GAGCGGCTGTCTCCACAAGT
RNP-F：CY5-TTCTGACCTGAAGGCTCTGCGCG-BHQ2
(4) sample gradient dilution
The new crown-pseudovirus with high concentration is mixed with DEPC water and then diluted by 10 times of gradient, and samples S1-S4 are obtained by 4 gradients (1E4 copies/mL-1E 7 copies/mL).
2. Nucleic acid release or extraction
(1) The nucleic acid releasing agents provided in examples 1 to 4 of the present application were equilibrated to room temperature and shaken up, and the following operations were performed for each example:
taking 6 PCR tubes, adding 10 mu L of the nucleic acid releasing agent provided by the application into each PCR tube, then respectively adding 30 mu L of each of the samples S1(1E7 copies/mL), S2(1E6 copies/mL), S3(1E5 copies/mL), S4(1E4 copies/mL), the negative quality control product and the positive quality control product, gently blowing, uniformly mixing without standing, wherein the whole process is about 1-3 min. Wherein the positive quality control product contains a 2019-nCoV detection fragment, and the negative reference product does not contain the 2019-nCoV detection fragment.
(2) Magnetic bead extraction was performed in accordance with the instructions for nucleic acid extraction or purification (magnetic bead method), and the following procedure was followed:
TABLE 3 extraction procedure for nucleic acid by the paramagnetic beads method
(3) The PCR reaction solution provided by the invention is respectively and directly added, and is used for standby after instantaneous centrifugation.
3. Fluorescent PCR procedure
The PCR procedure is as follows in table 4:
TABLE 4 PCR procedure
4. Analysis of results
The analysis results are shown in FIGS. 1 to 4 and FIGS. 5 to 8, wherein FIGS. 1 to 4 are schematic diagrams of PCR for detecting ORF1ab gene by magnetic bead method and nucleic acid releasing agent in examples 1 to 4, and FIGS. 5 to 8 are schematic diagrams of PCR for detecting N gene by magnetic bead method and nucleic acid releasing agent in examples 1 to 4, and it should be noted that only a part of the diagrams shown in the software are shown in FIGS. 1 to 4 and FIGS. 5 to 8, and the real experiment will automatically obtain detailed data.
The results of the sample measurements obtained in conjunction with FIGS. 1-4 and 2 are shown in tables 5-8 below:
TABLE 5 results of detection of sample by nucleic acid releasing agent and magnetic bead method in example 1
As can be seen from Table 5, the efficiency of nucleic acid releasing agent extraction in example 1 of the present application is equivalent to the magnetic bead method of nucleic acid extraction. Therefore, the extraction efficiency can be basically similar to that of the traditional magnetic bead method on the premise of simpler preparation method and operation steps.
TABLE 6 results of sample examination of the nucleic acid releasing agent of example 2 and the nucleic acid releasing agent of example 1
As can be seen from Table 6, the extraction efficiency of the nucleic acid releasing agent of example 2 of the present application is much lower than that of the nucleic acid releasing agent of example 1, and it can be seen that cysteine and NP-40 play a role in lysing cells and releasing nucleic acid in this protocol.
TABLE 7 results of sample examination of the nucleic acid releasing agent of example 3 and the nucleic acid releasing agent of example 1
It can be seen from table 7 that the extraction efficiency of the nucleic acid releasing agent of example 3 of the present application is much lower than that of the nucleic acid releasing agent of example 1, and cysteine functions to break down protein molecules and release nucleic acids in the present protocol.
TABLE 8 results of sample examination of the nucleic acid releasing agent of example 4 and the nucleic acid releasing agent of example 1
As can be seen from Table 8, the nucleic acid releasing agent of example 4 of the present application showed a much lower extraction efficiency than that of example 1, and NP-40 was found to function in this protocol to disrupt the nuclear membrane and release nucleic acids.
Example 7
This example, using the same design as example 6, still compares the extraction efficiency of the nucleic acid releasing agent of the present application with that of the conventional magnetic bead method. Except that this example 7 employs a rapid PCR procedure.
Nucleic acid releasing agent detection 2019-nCoV nucleic acid quality control product A (1E 7-1E 4 copies/mL) and magnetic bead method extraction nucleic acid detection 2019-nCoV nucleic acid quality control product B (1E 7-1E 4 copies/mL) rapid PCR program (iRapid 4).
1. Reagent preparation
(1) Nucleic acid releasing agent formulation of example 1:
the components of the nucleic acid releasing agent are shown in the table 1:
the preparation method of the nucleic acid releasing agent comprises the following steps: the components are measured according to the above table 1 and added in sequence, and the mixture is fully and evenly mixed to prepare the composition.
(2) Preparing a PCR reaction solution:
the addition amounts of the components of the PCR reaction solution are shown in Table 2:
wherein Fast Direct RT Premix Buffer II (dUTP) (Mg)2+free) (DG) contained (dATP, dTTP, dCTP, dGTP, dUTP), 3. mu.L of template was taken, and 25. mu.L of reaction system was used.
Wherein, the primer probe sequence is as follows:
ORF1ab-F：CCCTGTGGGTTTTACACTTAA
ORF1ab-R：ACGATTGTGCATCAGCTGA
ORF1ab-P：FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1
N-F：GGGGAACTTCTCCTGCTAGAAT
N-R：CAGACATTTTGCTCTCAAGCTG
N-P：VIC-TTGCTGCTGCTTGACAGATT–BHQ1
RNP-F：AGATTTGGACCTGCGAGCG
RNP-F：GAGCGGCTGTCTCCACAAGT
RNP-F：CY5-TTCTGACCTGAAGGCTCTGCGCG-BHQ2
(3) sample gradient dilution
The new crown-pseudovirus with high concentration is mixed with DEPC water and then diluted by 10 times of gradient, and samples S1-S4 are obtained by 4 gradients (1E4 copies/mL-1E 7 copies/mL).
2. Nucleic acid delivery
(1) The nucleic acid releasing agent in the embodiment 1 of the invention is balanced to room temperature and then shaken up, 6 PCR tubes are taken, 10 mu L of the nucleic acid releasing agent provided by the invention is added into each PCR tube, and then samples S1(1E7 copies/mL), S2(1E6 copies/mL), S3(1E5 copies/mL), S4(1E4 copies/mL), a negative quality control product and a positive quality control product are respectively added in turn by 30 mu L, and the samples are lightly blown and uniformly mixed. Wherein the positive quality control product contains a 2019-nCoV detection fragment, and the negative reference product does not contain the 2019-nCoV detection fragment.
(2) Magnetic bead extraction was performed exactly as described in the nucleic acid extraction or purification reagents (magnetic bead method), and the extraction procedure is shown in table 3:
(3) the PCR reaction solution provided by the invention is respectively and directly added, and is used for standby after instantaneous centrifugation.
3. Fluorescent PCR procedure
The PCR procedure is as follows in table 9:
table 9: PCR procedure
4. Analysis of results
As shown in FIGS. 9 to 13 and FIGS. 14 to 18, in which FIGS. 9 to 13 are schematic diagrams of the nucleic acid releasing agent and the magnetic bead method for detecting ORF1ab gene of examples 1 to 4 of the present invention (Rapid PCR), and FIGS. 14 to 18 are schematic diagrams of the nucleic acid releasing agent and the magnetic bead method for detecting N gene of examples 1 to 4 of the present invention (Rapid PCR), it is worth mentioning that FIGS. 9 to 13 and FIGS. 14 to 18 show only a part of the diagrams displayed in the software, and the real experiment will automatically obtain detailed data.
Summarizing the sample test results obtained in FIGS. 9-13 and FIGS. 14-18, the following Table 10 is shown:
TABLE 10 data table of results of magnetic bead assay and nucleic acid releasing agent assay in examples 1 to 4
The efficiency of nucleic acid releasing agent extraction of the present application is equivalent to magnetic bead extraction of nucleic acids, as analyzed by combining the data shown in FIGS. 9-13 and FIGS. 14-18 and Table 10. Therefore, the extraction efficiency can be basically similar to that of the traditional magnetic bead method on the premise of simpler preparation method and operation steps.
The nucleic acid releasing agent has the advantages of low consumption, low cost and safe use on the premise of easy discrimination, can be suitable for fluorescent quantitative PCR instruments of different types, particularly POCT, and can be widely applied to the fields of detection of pathogenic microorganisms, gene mutation detection, pet disease detection, forensic identification and the like.
The present invention is not described in detail in the prior art, and therefore, the present invention is not described in detail.
It is understood that the terms "a" and "an" should be interpreted as meaning that a number of one element or element is one in one embodiment, while a number of other elements is one in another embodiment, and the terms "a" and "an" should not be interpreted as limiting the number.
Although the use of the term in the present text is used more often, the possibility of using other terms is not excluded. These terms are used merely to more conveniently describe and explain the nature of the present invention; they are to be construed as being without limitation to any additional limitations that may be imposed by the spirit of the present invention.
The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as the present application, fall within the protection scope of the present invention.
Claims (9)
1. The nucleic acid releasing agent is characterized by comprising 0.5-1.5M Tris-HCl, 30-70mM EDTA, 0.5-1.5M cysteine, 10-30U/. mu.L RRI, DEPC water, NP-40 with the volume percentage concentration of 15-30% and Poly A with the volume percentage concentration of 3-7%, wherein the RRI is an RNase inhibitor.
2. The nucleic acid releasing agent of claim 1 consisting of 1M Tris-HCl, 50mM EDTA, 1M cysteine, 20U/. mu.L RRI, DEPC water, and NP-40 at 25% by volume, Poly A at 5% by volume.
3. The nucleic acid releasing agent of claim 1 wherein PolyA acts as a nucleic acid precipitating agent and cysteine is complexed with NP-40 as a non-ionic active agent to release nucleic acid.
4. A method for releasing nucleic acid, comprising the step of using the nucleic acid releasing agent according to any one of claims 1 to 3, comprising:
balancing the nucleic acid releasing agent to room temperature and shaking up;
adding the nucleic acid releasing agent after shaking up into a PCR reaction tube;
and adding a sample to be detected, blowing, beating and uniformly mixing to obtain the target nucleic acid, wherein the volume ratio of the nucleic acid releasing agent to the sample to be detected is 1: 3.
5. The method for releasing nucleic acid according to claim 4, wherein the sample to be tested comprises whole blood, serum, plasma or secretions.
6. The method for releasing nucleic acid according to claim 4, wherein the target nucleic acid is DNA or RNA.
7. The method for releasing nucleic acid according to claim 4, wherein the nucleic acid releasing agent is formulated as follows:
mu.L of 1M Tris-HCl, 0.4. mu.L of 50mM EDTA, 0.2. mu.L of cysteine, 0.2. mu.L of NP-40 with a volume percentage concentration of 25%, 0.2. mu.L of Poly A with a volume percentage concentration of 5%, 0.04. mu.L of 20U/. mu.L RRI and 7.96. mu.L of DEPC water are respectively and fully mixed to prepare the compound.
8. The method for releasing nucleic acid according to claim 4, wherein the method is applied to PCR detection of nucleic acid.
9. The method for releasing nucleic acid according to claim 4, wherein the release time of the nucleic acid releasing agent is 1 to 3 minutes.
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