CN113930418B - Nucleic acid releasing agent and nucleic acid releasing method thereof - Google Patents
Nucleic acid releasing agent and nucleic acid releasing method thereof Download PDFInfo
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Abstract
The invention relates to a nucleic acid releasing agent and a nucleic acid releasing method thereof, wherein the scheme comprises 0.5-1.5M Tris-HCl, 30-70mM EDTA, 0.5-1.5M cysteine, 10-30U/. Mu.L RRI, DEPC water, 15-30% NP-40 by volume percent concentration and 3-7% Poly A by volume percent concentration, wherein RRI is an RNase inhibitor. Balancing the nucleic acid releasing agent to room temperature and shaking uniformly; adding the nucleic acid releasing agent after shaking into a PCR reaction tube; and adding a sample to be detected, and blowing 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 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 medicine, in particular to a nucleic acid releasing agent and a nucleic acid releasing method thereof.
Background
PCR (Polymerase Chain Reaction ) is a molecular biological technique for amplifying specific nucleic acid fragments, and is characterized in that a trace amount of nucleic acid can be enriched and increased in a large amount, so that the aim of conveniently detecting the trace amount of nucleic acid is fulfilled. Among them, the PCR method commonly used in medical diagnosis is mainly a real-time fluorescent quantitative PCR (qPCR) method based on a double fluorescent probe, and targets for in vitro diagnosis using the qPCR method mainly include human genomic DNA, DNA viruses, bacteria, fungi, RNA viruses, and the like. Because the RNA has a single-chain structure, is unstable and is easy to degrade, the requirement on the sample treatment process is very high, and a complex method is required to perform pretreatment and nucleic acid extraction and purification on the RNA sample to be amplified, so that a stable result can be obtained by detecting pure nucleic acid.
For this reason, the current nucleic acid extraction methods applied to fluorescent quantitative PCR amplification mainly include the following 4 methods:
(1) Traditional boiling method: mixing the lysate with the blood sample, performing high-temperature pyrolysis, and centrifuging to obtain nucleic acid;
(2) Concentrating and boiling method: concentrating and precipitating virus nucleic acid by using polysaccharide, adding lysate to boil, and centrifuging to obtain nucleic acid;
(3) Centrifugal column method: adsorbing nucleic acid by a chromatographic column after cracking, and eluting to obtain purer nucleic acid;
(4) Magnetic bead method: adsorbing nucleic acid with magnetic bead, eluting to obtain purer nucleic acid.
The aforementioned first 2 extraction methods have disadvantages in that a plurality of steps such as transfer, pipetting, centrifugation are required, sample processing is time-consuming, and a part of nucleic acid is inevitably lost in the operation of high temperature processing, so that the transport amount of the nucleic acid sample finally used for PCR amplification is low. The centrifugal column method has a problem that the efficiency is low because the number of steps of manual operation is still large, although the purity of the nucleic acid extracted by the two boiling methods is greatly improved. The magnetic bead method has the advantages of simple extraction steps, high recovery rate, good purity and easy automation, but the product has high price and is not widely applied at present.
Therefore, there is a need for a nucleic acid releasing agent and a nucleic acid releasing method thereof which have high recovery efficiency, simple operation and low production cost.
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 achieve the above object, the present 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, 15-30% NP-40 and 3-7% Poly A by volume, wherein RRI is RNase inhibitor.
Working principle and beneficial effect: 1. compared with the prior art, the nucleic acid releasing agent is completely different from the traditional nucleic acid releasing agent in composition, the application uses PolyA as a nucleic acid sedimentation aid, and cysteine and NP-40 are used as nonionic active agents to be matched with the PolyA to release nucleic acid of a sample to be tested, specifically, the cysteine and the NP-40 are in the scheme: the two are used as nonionic surfactants, can lyse cell membranes to enable nucleic acid to be released rapidly, and in addition, NP-40 can protect reverse transcriptase to enable reverse transcriptase to work normally in alkaline environment, so that the use of the reverse transcriptase does not need to use lysate to boil, and the use of a centrifugal column method and a magnetic bead method to extract nucleic acid is also not needed, and only the nucleic acid can be obtained by mixing the nucleic acid with a sample to be detected in a PCR reaction tube, so that the operation difficulty can be remarkably reduced, the operation time can be shortened, the waste of nucleic acid can be reduced, and the advantages of high extraction efficiency, simplicity in operation can be achieved, and sample treatment and low production cost can be completed within 3 minutes at room temperature;
2. the nucleic acid released by 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 also needs to be carried out by matching a nucleic acid extractor and a magnetic frame, the general running time of a program is about 30 minutes, the cost is high, and the time is long.
3. The preparation method of the nucleic acid releasing agent is extremely simple, and the preparation method can be realized by uniformly mixing and shaking the above various costs, thereby remarkably reducing the production cost and the preparation difficulty.
Further, it consists of 1M Tris-HCl, 50mM EDTA, 1M cysteine, 20U/. Mu.L RRI, DEPC water, NP-40 at a concentration of 25% by volume, poly A at a concentration of 5% by volume.
The nucleic acid releasing method adopts the nucleic acid releasing agent and specifically comprises the following steps:
balancing the nucleic acid releasing agent to room temperature and shaking uniformly;
adding the nucleic acid releasing agent after shaking into a PCR reaction tube;
and adding a sample to be detected, and blowing 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. Compared with the prior art, the method has the advantages that sample nucleic acid does not need to be extracted independently, the target nucleic acid can be obtained 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, the operations such as centrifugation, supernatant discarding, tube rotating and the like in the prior art are thoroughly omitted, the experimental steps are greatly simplified, and the operations such as boiling, transferring reaction vessels and the like are not needed, so that the nucleic acid is not lost, 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.
The nucleic acid releasing agent can be used for rapidly and accurately measuring the nucleic acid content 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 preparation method of the nucleic acid releasing agent comprises the following steps:
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 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 taken and fully mixed to prepare the drug.
The arrangement proves that the dosage of the nucleic acid releasing agent is small, the cost can be kept low on the premise of easy discrimination, the use is safe, and the nucleic acid releasing agent can be suitable for fluorescent quantitative PCR instruments of different types, especially POCT (point of care testing), 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 diagram showing the detection of ORF1ab gene by the nucleic acid releasing agent and magnetic bead method of example 1 of the present invention;
FIG. 2 is a schematic diagram showing the 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 showing the 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 showing the 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 diagram of the detection of N gene by the nucleic acid releasing agent and magnetic bead method of example 1;
FIG. 6 is a schematic diagram showing 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 diagram showing 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 diagram showing 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 the detection of ORF1ab gene (rapid PCR) of example 1;
FIG. 11 is a schematic diagram of the detection of ORF1ab gene (rapid PCR) of example 2;
FIG. 12 is a schematic diagram of the detection of ORF1ab gene (rapid PCR) of example 3;
FIG. 13 is a schematic diagram of the detection of ORF1ab gene (rapid PCR) of example 4;
FIG. 14 is a schematic diagram of detection of N gene by magnetic bead method (rapid PCR);
FIG. 15 is a schematic diagram of detection of the N gene in example 1 (rapid PCR);
FIG. 16 is a schematic diagram of detection of the N gene in example 2 (rapid PCR);
FIG. 17 is a schematic diagram of detection of N gene in example 3 (rapid PCR);
FIG. 18 is a schematic diagram of detection of N gene in example 4 (Rapid PCR).
Detailed Description
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 are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the 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% and Poly A with the volume percentage concentration of 5%.
The preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
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 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 taken and fully mixed to prepare the drug.
Wherein, 1M Tris-HCl is 1mol/L Tris hydrochloride; 50mM EDTA is ethylenediamine tetraacetic acid at 50 mmol/L; RRI is an rnase inhibitor; DEPC water is ultrapure water (primary water) treated with DEPC (diethyl pyrocarbonate ) and sterilized at high temperature and high pressure, colorless liquid, and contains no RNA, DNA and protein as impurities; NP-40 is an abbreviation for Nonidet P40, chinese translated into ethylphenyl polyethylene glycol, a mild nonionic detergent; poly A acts as a nucleic acid precipitation aid.
Example 2: blank 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.
The preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
mu.L of 1M Tris-HCl, 0.4 mu.L of 50mM EDTA, 0.2 mu.L of Poly A with a volume percentage concentration of 5%, 0.04 mu.L of 20U/. Mu.L RRI and 8.36 mu.L of DEPC water are taken and fully mixed to prepare the drug.
Example 3: control experiments with cysteine replaced with sodium lauroyl sarcosinate
The nucleic acid releasing agent consists of 1M Tris-HCl, 50mM EDTA, 1M sodium lauroyl sarcosinate, 20U/. Mu.LRRI, DEPC water, NP-40 with the volume percentage concentration of 25% and Poly A with the volume percentage concentration of 5%.
The preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
mu.L of 1M Tris-HCl, 0.4 mu.L of 50mM EDTA, 0.2 mu.L of 1M sodium lauroyl sarcosinate, 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 taken and fully mixed to prepare the drug.
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 and 5% Poly A.
The preparation method of the nucleic acid releasing agent for one experiment comprises the following steps:
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 Triton X-100 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 taken and fully mixed to prepare the drug.
It is worth noting that the configuration environments and conditions of the above embodiments 1 to 4 are completely identical.
Example 5
The nucleic acid release method using the nucleic acid release agent of the above examples 1 to 4 specifically comprises the following steps:
step 1, balancing the nucleic acid releasing agent to room temperature and shaking uniformly;
step 2, adding the nucleic acid releasing agent after shaking to a PCR reaction tube;
and step 3, adding a sample to be tested, and blowing and uniformly mixing to obtain target nucleic acid, wherein the volume ratio of the nucleic acid releasing agent to the sample to be tested is 1:3, and the sample to be tested 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 composition, and the method uses PolyA as a nucleic acid sedimentation aid, uses cysteine and NP-40 as nonionic active agents to be matched with PolyA to release nucleic acid of a sample to be detected, is similar to the magnetic bead method for extracting the nucleic acid, but is completely different in operation method, so that the method does not need to use lysate to boil and does not need to adopt a centrifugal column method and a magnetic bead method for extracting the nucleic acid when in use, and the nucleic acid can be obtained only by mixing the nucleic acid with the sample to be detected in a PCR reaction tube, so that the operation difficulty can be remarkably reduced, the operation time can be shortened, and the waste of the nucleic acid can be reduced, thereby achieving the advantages of high extraction efficiency, simplicity in operation and low production cost.
Example 6
This example is a practical application of examples 1-4, and the effectiveness of the nucleic acid releasing agent and the nucleic acid releasing method of the present application was verified by a common PCR procedure (LC 480) by comparing the nucleic acid releasing agent of examples 1-4 with the nucleic acid extracted by the conventional magnetic bead method.
The instrument used in the experiment is an LC480 real-time fluorescence quantitative PCR instrument, which is purchased from Roche diagnostics Inc.; the iRapid4 real-time fluorescent quantitative PCR instrument is manufactured by Hangzhou dean biotechnology Co., ltd; nucleic acid extractor EB1000 is manufactured by hangzhou dean biotechnology limited;
the nucleic acid releasing agent and the nucleic acid extracting or purifying agent (magnetic bead method) are produced by Di an biotechnology Co., ltd., hangzhou, the purity of the agent is molecular biology grade, the agent and the enzyme system used in the PCR reaction solution are purchased from Shanghai Bai Biotechnology Co., ltd., and the primer and the probe are synthesized by Shanghai Bai Ge biotechnology Co., ltd.; new coronaviruses were custom synthesized by the Biotechnology Co., ltd. In Paul and reported for quality inspection.
Nucleic acid releasing agent detection 2019-nCoV nucleic acid quality control A (1E 7-1E 4 copies/mL) and magnetic bead method extraction nucleic acid detection 2019-nCoV nucleic acid quality control B (1E 7-1E 4 copies/mL).
1. Reagent preparation
(1) Example 1-example 4 nucleic acid delivery agent formulation:
the components of the nucleic acid delivery agent provided in example 1 are shown in Table 1 below:
TABLE 1 nucleic acid Release Agents provided in example 1
| Reagent name | Volume (everyone) |
| 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 table 1, added in sequence and fully and uniformly mixed.
The formulation of the nucleic acid releasing agent of examples 2 to 4 is the same as that described in the above examples, and the description thereof is not repeated here.
(2) Nucleic acid extraction or purification reagent (magnetic bead method) configuration:
the nucleic acid extraction or purification reagent is produced by Hangzhou dean biotechnology Co-Ltd, and the product mainly comprises a lysate, a washing solution I, a washing solution II, a washing solution III, magnetic bead suspension, an eluent, proteinase 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 liquid are shown in the following Table 2:
table 2: PCR reaction liquid component
Wherein Fast Direct RT Premix Buffer II (dUTP) (Mg2+free) (DG) contained (dATP, dTTP, dCTP, dGTP, dUTP), 3. Mu.L of the template was used, and the reaction system was 25. Mu.L.
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 high concentration of the novel coronavirus was mixed with DEPC water and subjected to 10-fold gradient dilution for a total of 4 gradients (1E 4 copies/mL to 1E7 copies/mL) to obtain samples S1 to S4.
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 then shaken well, and the following procedure was carried out 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, and sequentially adding samples S1 (1E 7 copies/mL), S2 (1E 6 copies/mL), S3 (1E 5 copies/mL), S4 (1E 4 copies/mL), negative quality control substances and positive quality control substances respectively in 30 mu L, gently blowing and mixing uniformly, and standing is not needed, wherein the whole process is approximately 1-3min. Wherein the positive quality control contains 2019-nCoV detection fragments and the negative reference does not contain 2019-nCoV detection fragments.
(2) Magnetic bead extraction was performed with strict reference to the nucleic acid extraction or purification reagents (magnetic bead method) instructions, and the extraction procedure is as follows in table 3:
TABLE 3 extraction procedure for extracting nucleic acid by magnetic bead method
(3) The PCR reaction liquid provided by the invention is directly added respectively, and is used after instantaneous centrifugation.
3. Fluorescent PCR program
The PCR procedure is as follows table 4:
TABLE 4 PCR procedure
4. Analysis of results
The analysis results are shown in FIGS. 1 to 4 and 5 to 8, wherein FIGS. 1 to 4 are schematic diagrams of PCR for detecting ORF1ab gene by the magnetic bead method and the nucleic acid releasing agents of examples 1 to 4, and FIGS. 5 to 8 are schematic diagrams of PCR for detecting N gene by the magnetic bead method and the nucleic acid releasing agents of examples 1 to 4, and it is noted that only a part of the diagrams shown in the software are shown in FIGS. 1 to 4 and 5 to 8, and detailed data are automatically obtained in the real experiment.
The sample test results obtained in connection with fig. 1-4 and fig. 2 are shown in tables 5-8 below:
TABLE 5 sample detection results of the nucleic acid releasing agent and magnetic bead method of example 1
It can be seen from Table 5 that the extraction efficiency of the nucleic acid releasing agent of example 1 of the present application is equivalent to that of the magnetic bead method. 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 sample detection results of the nucleic acid releasing agent of example 2 and the nucleic acid releasing agent of example 1
It can be seen from Table 6 that the nucleic acid releasing agent of example 2 of the present application has much lower extraction efficiency than the nucleic acid releasing agent of example 1, and that cysteine and NP-40 act to lyse cells and release nucleic acid in this scheme.
TABLE 7 sample detection results 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 nucleic acid releasing agent of example 3 of the present application has much lower extraction efficiency than the nucleic acid releasing agent of example 1, and that cysteine plays a role in destroying protein molecules and releasing nucleic acids in this scheme.
TABLE 8 sample detection results of the nucleic acid delivery agent of example 4 and the nucleic acid delivery agent of example 1
It can be seen from Table 8 that the nucleic acid releasing agent of example 4 of the present application has an extraction efficiency far lower than that of the nucleic acid releasing agent of example 1, and that NP-40 plays a role in disrupting the nuclear membrane and releasing nucleic acid in this scheme.
Example 7
This example uses the same design as example 6, yet still compares the extraction efficiency of the nucleic acid releasing agent of this application with that of the conventional magnetic bead method. Except that this example 7 employed a rapid PCR procedure.
Nucleic acid releasing agent detection 2019-nCoV nucleic acid quality control A (1E 7-1E 4 copies/mL) and magnetic bead method extraction nucleic acid detection 2019-nCoV nucleic acid quality control B (1E 7-1E 4 copies/mL) rapid PCR procedure (iRapid 4).
1. Reagent preparation
(1) Preparation of nucleic acid releasing agent of example 1:
the nucleic acid releasing agent comprises the following components in table 1:
the preparation method of the nucleic acid releasing agent comprises the following steps: the components are measured according to the table 1, added in sequence and fully and uniformly mixed.
(2) Preparing a PCR reaction solution:
the addition amounts of the components of the PCR reaction liquid are shown in Table 2:
therein Fast Direct RT Premix Buffer II (dUTP) (Mg 2+ free) (DG) contains (dATP, dTTP, dCTP, dGTP, dUTP), 3. Mu.L of the template is used, and the reaction system is 25. Mu.L.
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 high concentration of the novel coronavirus was mixed with DEPC water and subjected to 10-fold gradient dilution for a total of 4 gradients (1E 4 copies/mL to 1E7 copies/mL) to obtain samples S1 to S4.
2. Nucleic acid release
(1) The nucleic acid releasing agent of the embodiment 1 is balanced to room temperature, and then the mixture is shaken uniformly, 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 samples S1 (1E 7 copies/mL), S2 (1E 6 copies/mL), S3 (1E 5 copies/mL) and S4 (1E 4 copies/mL) are respectively added into each PCR tube in sequence, and 30 mu L of each PCR tube is gently blown and mixed uniformly. Wherein the positive quality control contains 2019-nCoV detection fragments and the negative reference does not contain 2019-nCoV detection fragments.
(2) Magnetic bead extraction was performed with strict reference to the nucleic acid extraction or purification reagents (magnetic bead method) instructions, and the extraction procedure is as follows in table 3:
(3) The PCR reaction liquid provided by the invention is directly added respectively, and is used after instantaneous centrifugation.
3. Fluorescent PCR program
The PCR procedure is as follows table 9:
table 9: PCR program
4. Analysis of results
FIGS. 9 to 13 and 14 to 18 show the nucleic acid releasing agent and the magnetic bead method of examples 1 to 4 of the present invention (rapid PCR), and FIGS. 14 to 18 show the nucleic acid releasing agent and the magnetic bead method of examples 1 to 4 of the present invention (rapid PCR) for detecting the N gene, and it is noted that FIGS. 9 to 13 and 14 to 18 show only a part of the diagrams shown in the software, and that the real experiment automatically gives detailed data.
The sample test results obtained in the summary of fig. 9-13 and fig. 14-18 are shown in table 10 below:
TABLE 10 magnetic bead method and data table for detection results of nucleic acid releasing agent of examples 1 to 4
In connection with the data analysis as in FIGS. 9-13 and 14-18 and Table 10, the nucleic acid releasing agent extraction efficiency of the present application is equivalent to that of the magnetic bead method for extracting nucleic acid. 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 application has the advantages that the dosage of the nucleic acid releasing agent is small, the cost is low, the use is safe on the premise of easy discrimination, the application is applicable to fluorescent quantitative PCR instruments of different types, particularly POCT (point of care testing), and the application can be widely applied to the fields of detection of pathogenic microorganisms, gene mutation detection, pet disease detection, forensic identification and the like.
The invention is not described in detail in the prior art, and therefore, the invention is not described in detail.
It will be understood that the terms "a" and "an" should be interpreted as referring to "at least one" or "one or more," i.e., in one embodiment, the number of elements may be one, while in another embodiment, the number of elements may be plural, and the term "a" should not be interpreted as limiting the number.
Although specific terms are used more herein, the use of other terms is not precluded. These terms are used merely for convenience in describing and explaining the nature of the invention; they are to be interpreted as any additional limitation that is not inconsistent with the spirit of the present invention.
The present invention is not limited to the above-mentioned preferred embodiments, and any person can obtain various other products without departing from the scope of the present invention, but any changes in shape or structure are within the scope of the present invention, which is the same or similar to the present invention.
Claims (7)
1. The nucleic acid releasing agent is characterized by being prepared from 0.5-1.5M Tris-HCl, 30-70mM EDTA, 0.5-1.5M cysteine, 10-30U/. Mu.L RRI, DEPC water, 15-30% NP-40 and 3-7% Poly A by volume percent, wherein RRI is an RNase inhibitor;
wherein, polyA is used as nucleic acid sedimentation aid, cysteine and NP-40 are used as nonionic active agent to match with PolyA to release nucleic acid;
the preparation method of the nucleic acid releasing agent comprises the following steps:
mu.L of the Tris-HCl, 0.4 mu.L of the EDTA, 0.2 mu.L of the cysteine, 0.2 mu.L of the NP-40, 0.2 mu.L of the Poly A, 0.04 mu.L of the RRI and 7.96 mu.L of the DEPC water were thoroughly mixed to prepare the drug.
2. The nucleic acid delivery agent of claim 1, wherein the agent is formulated from 1M Tris-HCl, 50mM EDTA, 1M cysteine, 20U/. Mu.L RRI, DEPC water, and 25% NP-40 by volume and 5% Poly A by volume.
3. The method for releasing nucleic acid, characterized by using the nucleic acid releasing agent according to claim 1 or 2, comprising the steps of:
balancing the nucleic acid releasing agent to room temperature and shaking uniformly;
adding the nucleic acid releasing agent after shaking into a PCR reaction tube;
and adding a sample to be detected, and blowing 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.
4. The method of claim 3, wherein the sample to be tested comprises whole blood, serum, plasma or secretions.
5. The method for releasing nucleic acid according to claim 3, wherein the target nucleic acid is DNA or RNA.
6. The method for releasing nucleic acid according to claim 3, wherein the method is applied to PCR detection of nucleic acid.
7. The method for releasing nucleic acid according to claim 3, wherein the time for releasing the nucleic acid releasing agent is 1 to 3 minutes.
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