CN112813140A - 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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- CN112813140A CN112813140A CN202110199917.9A CN202110199917A CN112813140A CN 112813140 A CN112813140 A CN 112813140A CN 202110199917 A CN202110199917 A CN 202110199917A CN 112813140 A CN112813140 A CN 112813140A
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Abstract
The invention relates to the technical field of molecular biology, in particular to a nucleic acid releasing agent, a method for rapidly releasing nucleic acid and application thereof. The nucleic acid releasing agent consists of 20-100 mM Tris-HCl, SDS with the mass/volume ratio of 0.01-0.5%, triton 100 with the volume ratio of 0.04-0.4%, 10-500mM ammonium sulfate and betaine with the mass/volume ratio of 0.01-2%, and comprises the following specific steps: adding samples from various sample sources into the nucleic acid releasing agent in a proper manner, then oscillating and uniformly mixing, and standing at room temperature for 1min to absorb supernatant so as to obtain released nucleic acid. The nucleic acid releasing agent of the invention has the following advantages: firstly, the method comprises the following steps: the nucleic acid in serum, plasma, urine, saliva, buccal swab, dry blood spot, forensic sample and environmental sample can be rapidly released, and the nucleic acid can be used for downstream experiments such as detection, cloning, sequence analysis, molecular hybridization and the like; II, secondly: can inactivate viruses or other pathogenic microorganisms in a sample, particularly a swab sample, ensure the biological safety of test operators, greatly shorten the nucleic acid extraction and purification time, avoid the recovery step of nucleic acid, avoid the loss or pollution of nucleic acid and have the advantage of simple operation.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a nucleic acid releasing agent and a nucleic acid releasing method thereof.
Background
Respiratory Tract Infection (RTI) is the most common type of disease in humans and is one of the leading causes of morbidity and mortality in the population worldwide. It has been demonstrated that most respiratory diseases are caused by viruses. Viral infections are potentially extremely harmful, for example the novel coronavirus (SARS-CoV-2), a type of coronavirus not previously found in humans, which causes acute infectious pneumonia (COVID-19), and the initial symptoms of the infected are fever, weakness and dry cough. With subsequent progressive dyspnea, severe cases may develop acute respiratory distress syndrome or septic shock, and even death.
The gold standard of viral infection is real-time fluorescence quantitative PCR, is a molecular diagnosis method for exponentially amplifying specific nucleic acid fragments and judging whether a target to be detected exists or not by using a fluorescence signal, and is 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. Because the structure of RNA is single-stranded, it is unstable and easy to degrade, and it needs more complex method to pre-treat the amplified RNA sample and extract and purify the nucleic acid, and after obtaining pure nucleic acid, it can detect to obtain stable result. In the current clinical sample detection, the magnetic bead method has simple extraction steps, high recovery efficiency and easy automatic extraction, but the extraction and purification of nucleic acid in a sample occupy a certain time, and special matched automatic extraction equipment and related consumables are needed, so that the speed of sample detection and field application are seriously influenced under the condition of large sample amount. Meanwhile, the pretreatment of the pathogen sample is also the most dangerous link, and generally, after workers need to strictly perform three-level biosafety protection equipment in a sample preparation area, the relevant experiment of nucleic acid extraction is completed in a biosafety cabinet, so that not only is virus infection prevented, but also the sample is prevented from being polluted, and the result is inaccurate. Therefore, the development of a method for rapidly releasing nucleic acid, which can rapidly release nucleic acid, is not easy to cause cross contamination, and has no influence on the subsequent nucleic acid detection experiment, is a problem to be solved.
Disclosure of Invention
Aiming at the problems in the prior art of pretreatment of nucleic acid detection samples, the invention provides a nucleic acid releasing agent, a method for quickly releasing nucleic acid and application thereof, aiming at greatly shortening the nucleic acid extraction pure time, simplifying the steps of pretreatment of samples in nucleic acid detection and obviously improving the detection flux.
The nucleic acid releasing agent consists of 20-100 mM Tris-HCl, SDS with the mass/volume ratio of 0.01-0.5%, triton 100 with the volume ratio of 0.04-0.4%, 10-500mM ammonium sulfate and DTT with the mass/volume ratio of 0.01-2%;
wherein the pH value of Tris-HCl is 6.5-8.0.
The method for rapidly releasing nucleic acid of the invention is carried out according to the following steps:
(1) mixing different types of samples with the nucleic acid releasing agent according to a certain proportion, and carrying out vortex oscillation;
(2) and (3) incubating for a certain time, wherein the incubation can be carried out at normal temperature for 5-10 min, the preferable heating temperature is 70-90 ℃, the incubation time is 2min, and the nucleic acid can be effectively released.
Compared with the prior art, the invention has the following beneficial effects:
1. the protein denaturant with strong nucleic acid releaser can quickly break protein structures of cells, viruses and the like; the virus and other pathogenic microorganisms in the sample can be inactivated quickly, and the safety is high;
2. the protein denaturant in the nucleic acid releaser can completely inhibit the activity of Dnase and Rnase, can protect nucleic acid at normal temperature or even high temperature, avoids the degradation of nucleic acid, especially RNA, and does not need to be transported at low temperature;
3. the nucleic acid release method can quickly release the nucleic acid in serum, plasma, urine, saliva, oral swabs, dry blood spots, forensic samples and environmental samples, and the released nucleic acid can be used for downstream experiments such as detection, cloning, sequence analysis, molecular hybridization and the like;
4. the nucleic acid release method greatly shortens the nucleic acid extraction pure time, simplifies the steps of sample pretreatment in nucleic acid detection and obviously improves the detection flux.
Drawings
FIG. 1 is a fluorescent PCR assay of a buccal swab sample extracted with a nucleic acid releasing agent of the invention: the internal references of 80 samples are all amplified normally;
FIG. 2 is a fluorescence PCR assay of buccal swab samples extracted with the nucleic acid releasing agent of the present invention, wherein 3 target genes in the positive reference and the strong positive reference are normally amplified;
FIG. 3 is a graph of fluorescent PCR detection of buccal swab samples of different pseudovirus copy numbers extracted with nucleic acid releasing agents of the invention: the amplification of the sample is linearly distributed, and the 100 copy number can be detected;
FIG. 4 is a sample of pseudovirus preserved and extracted at 37 ℃ with the nucleic acid releasing agent of the present invention: the pseudovirus RNA can be stably stored in the nucleic acid releasing agent;
FIG. 5 is a diagram showing the treatment of a blood sample with a nucleic acid releasing agent of the present invention and the release of blood virus DNA: indicating that the nucleic acid releasing agent can stably extract virus DNA from blood.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The technical solution of the present invention is further described in detail below with reference to specific examples.
Example 1 detection of respiratory viruses
(1) The throat swab gently scrapes human epidermal mucosal cells, and the cotton ball part of the swab is infiltrated into the nucleic acid releasing agent; after the cotton balls are completely soaked, covering the pipe cover tightly, reversing the pipe cover up and down, uniformly mixing for 90 seconds, and then performing instantaneous centrifugation;
(2) heating at 90 ℃ for 2min, sucking 10 microliters of supernatant to obtain released nucleic acid, and performing fluorescence RT-PCR detection;
(3) in this example, the specific amplification primer pair for amplifying the ORF1 region of the viral sequence was the following sequence:
ORF1a-F：TGCCGTTGCCACATAGAT(SEQ NO.1)
ORF1a-R：GGTCATTAGCACAAGTTGTAGG(SEQ NO.2)
the specific fluorescent probe for amplifying the ORF1 region of the virus sequence has the following sequences:
ORF1a-P：ATCAGGAGATGC CACAACTGC(SEQ NO.3)
the specific primer pair for amplifying the virus N protein sequence has the following sequences:
ORFN-F：ATGTACTCATTCGTTTCGGAAG(SEQ NO.4)
ORFN-R：ATACCACGAAAGCAAGAAAAAG(SEQ NO.5)
the specific fluorescent probe for amplifying the virus N protein sequence has the following sequence:
ORFN-P：CTGCTCTTGCTTTGCTGCTGCTTGAC(SEQ NO.6)
the specific amplification primer pair of the endogenous reference gene RnaseP for monitoring the detection process has the following sequence:
RNaseP-F：GTGTGTCCACTCAGGCTTGT(SEQ NO.7)
RNaseP-R：AGATGGGTCTCAGGTGCAG(SEQ NO.8)
the specific fluorescent probe of the endogenous reference gene RnaseP for monitoring the detection process has the following sequence:
RNaseP-P：TTTCTCAGATCAGCATTTGCTGGC(SEQ NO.9)
the working concentration of the primer pair and the probe is 20 mu M (20 mu mol/L);
(4) RT-PCR reaction reagent: contains random primer with 6N basic groups, dNTP, Tris-HCl, NaCl, KCl, DTT, reverse transcriptase, ATP, MgCl2, hot start Taq enzyme and the like;
(5) the results are shown in figure 1 and figure 2, wherein figure 1 shows that the internal references of the sample are amplified well, and figure 2 shows that the weak positive reference and the strong positive reference are amplified.
Example 2
(1) Adding 100, 1000 and 10000 COVID-19 pseudoviruses into 200 microliters of nucleic acid releasing agent, reversing the mixture up and down, uniformly mixing the mixture for 90 seconds, and then performing instantaneous centrifugation;
(2) heating at 90 ℃ for 2min, sucking 10 microliters of supernatant to obtain released nucleic acid, and performing fluorescence RT-PCR detection;
(3) the results are shown in FIG. 3, which shows that the amplification of the sample is linearly distributed and 100 copies can be detected.
Example 3
(1) Add 2x105Adding the COVID-19 pseudovirus into 10mL of nucleic acid releasing agent, turning upside down, mixing uniformly for 90 seconds, centrifuging instantaneously, taking 50 microliters of the COVID-19 pseudovirus, and placing the mixture at 37 ℃ for 24, 48, 72, 96, 120, 144, 168 and 192 hours;
(2) heating at 90 ℃ for 2min, sucking 10 microliters of supernatant to obtain released nucleic acid, and performing fluorescence RT-PCR detection;
(3) the results are shown in FIG. 4, and the specific ct values are shown in Table 1, which shows that the pseudoviral RNA can be stably preserved in the nucleic acid releasing agent.
Example 4
(1) Adding 50 mu L of original whole blood sample of the swine fever, a diluted sample of 100 times and a diluted sample of 10000 times into 200 mu L of nucleic acid releasing agent, centrifuging, removing supernatant, reversing the upside down, uniformly mixing for 90 seconds, and then performing instantaneous centrifugation;
(2) heating at 90 deg.C for 2min, sucking 10 microliters of supernatant to obtain released nucleic acid, and performing fluorescence PCR detection;
(3) the results are shown in FIG. 5, which shows that the nucleic acid releasing agent can stably extract viral DNA from blood.
Claims (8)
1. A nucleic acid releasing agent is characterized by consisting of 20-100 mM Tris-HCl, SDS with the mass/volume ratio of 0.01-0.5%, triton 100 with the volume ratio of 0.04-0.4%, 10-500mM ammonium sulfate and DTT with the mass/volume ratio of 0.01-2%.
2. A nucleic acid releasing agent according to claim 1, characterized in that Tris-HCl has a pH of 6.5 to 8.0.
3. A method for rapidly releasing nucleic acid using the nucleic acid releasing agent of claim 1, wherein: the sample types are swab samples (pharynx swab/nose swab/anus swab), artificially synthesized pseudoviruses, in-vitro transcription RNA, saliva, alveolar lavage fluid and other samples.
4. A method for rapidly releasing nucleic acid using the nucleic acid releasing agent of claim 1, wherein the pretreatment procedure for a throat swab, a nasal swab or an anal swab sample is as follows: breaking the swab head after sampling, immersing the swab head in a sampling tube containing a sample releasing agent, and screwing the tube cover.
5. A method for quickly releasing nucleic acid by using the nucleic acid releasing agent as claimed in claim 1 features that the pretreatment procedure of in vitro transcription of RNA specimen for artificially synthesized pseudovirus includes such steps as taking a proper quantity of specimen, diluting to a certain volume of Buffer PBS/specimen releasing agent, and mixing.
6. A method for rapidly releasing nucleic acid using the nucleic acid releasing agent of claim 1, wherein the pretreatment procedure for a sample of alveolar lavage fluid for saliva comprises the steps of taking a sample of a predetermined amount of the nucleic acid releasing agent and mixing the sample with the nucleic acid releasing agent in a volume ratio of 1: 1 and mixing.
7. A method for rapidly releasing nucleic acid by using the nucleic acid releasing agent as claimed in claim 3, characterized in that after a sample is uniformly mixed with the nucleic acid releasing agent, the mixture is shaken and uniformly mixed for 30 seconds, kept stand for 2min, and a proper volume of supernatant is sucked and added into a PCR amplification system for amplification detection.
8. A method for rapidly releasing nucleic acid by using the nucleic acid releasing agent of claim 7, wherein the method is better when the nucleic acid is heated at 90 ℃ for 2min after being uniformly stirred for 30 seconds.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528508A (en) * | 2021-08-06 | 2021-10-22 | 广州氧颜医疗科技有限公司 | Nucleic acid releasing agent and nucleic acid releasing method |
| CN113621718A (en) * | 2021-08-09 | 2021-11-09 | 浙江舟鲜生食品科技有限公司 | Method for rapidly detecting salmonella in food |
| CN114672541A (en) * | 2022-03-21 | 2022-06-28 | 生工生物工程（上海）股份有限公司 | Nucleic acid release solution, kit and nucleic acid release method |
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2021
- 2021-02-23 CN CN202110199917.9A patent/CN112813140A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528508A (en) * | 2021-08-06 | 2021-10-22 | 广州氧颜医疗科技有限公司 | Nucleic acid releasing agent and nucleic acid releasing method |
| CN113621718A (en) * | 2021-08-09 | 2021-11-09 | 浙江舟鲜生食品科技有限公司 | Method for rapidly detecting salmonella in food |
| CN114672541A (en) * | 2022-03-21 | 2022-06-28 | 生工生物工程（上海）股份有限公司 | Nucleic acid release solution, kit and nucleic acid release method |
| CN114672541B (en) * | 2022-03-21 | 2024-02-02 | 生工生物工程（上海）股份有限公司 | Nucleic acid release liquid, kit and nucleic acid release method |
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