CN112501159A - Universal free nucleic acid protective agent - Google Patents
Universal free nucleic acid protective agent Download PDFInfo
- Publication number
- CN112501159A CN112501159A CN202011414505.4A CN202011414505A CN112501159A CN 112501159 A CN112501159 A CN 112501159A CN 202011414505 A CN202011414505 A CN 202011414505A CN 112501159 A CN112501159 A CN 112501159A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- protective agent
- free nucleic
- sample
- tube
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a universal free nucleic acid protective agent. The protective agent comprises the following components: 2-200mM of ethylenediamine tetraacetic acid, 5-20mM of Tris-HCl, 0.02-2M of dithiothreitol, 0.1-30mg/mL of glycine, 0.1-30mg/mL of glutamine, 0.1-20mg/mL of cysteine, 0.2-1M of trehalose, 0.1-20mg/mL of sodium fluoride, 0.2-30mg/mL of sodium hydroxymethyl glycinate and 0.02-0.5mM of diethyl pyrocarbonate, and adjusting the pH value to 5-7. The protective agent can protect both free DNA and free RNA, prevent the breakage of nucleated cells and protect a sample from being stored at normal temperature.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a universal free nucleic acid protective agent.
Background
Free nucleic acid is nucleic acid existing outside cells in a Free state, including Free DNA (cfDNA) and Free RNA (cfRNA), and is widely present in serum, plasma, cerebrospinal fluid, urine, saliva or feces of animals, plants and humans. The source of the nucleic acid mainly comprises nucleic acid released by normal apoptosis, nucleic acid released into blood by necrotic tissues, particularly tumor tissues, and nucleic acid infected by viruses and bacteria. Under the background of precise medicine, the free nucleic acid is an important clinical noninvasive molecular marker, has great clinical application value along with the development of gene diagnosis technology, and has important significance in early diagnosis, staging, treatment monitoring, prognosis judgment, prenatal gene diagnosis, pathogen infection gene detection and other aspects. The content of free nucleic acid is generally low, so that the degradation of the free nucleic acid in a sample is prevented, and the increase of nucleic acid background caused by the breakage of nucleated cells is avoided, so that the reduction of detection sensitivity and false negative of an experimental result are avoided, and a new demand is provided for the collection and storage of the sample.
In the clinical process, due to the limitation of conditions or time, the clinical sample cannot be detected at the first time or needs to be detected after transportation, the clinical sample needs to be protected, the molecular markers such as free nucleic acid in the sample are protected from being affected, and the lysis of nucleated cells is prevented. There is conventional heparin tube on the market mainly has the function of preventing the blood coagulation, can not play the effect of protection free nucleic acid, and free nucleic acid heparin tube on the market is mainly the protection free DNA, rarely has free DNA and free RNA blood sampling tube of protection altogether, and for foreign import moreover, the cost is higher, does not have the general sampling tube of many samples more.
Disclosure of Invention
The invention aims to solve the technical problem of providing a universal free nucleic acid protective agent to fill the blank of the prior art.
The invention provides a universal free nucleic acid protective agent, which comprises the following components: 2-200mM of ethylenediamine tetraacetic acid, 5-20mM of Tris-HCl, 0.02-2M of dithiothreitol, 0.1-30mg/mL of glycine, 0.1-30mg/mL of glutamine, 0.1-20mg/mL of cysteine, 0.2-1M of trehalose, 0.1-20mg/mL of sodium fluoride, 0.2-30mg/mL of sodium hydroxymethyl glycinate and 0.02-0.5mM of diethyl pyrocarbonate, and adjusting the pH value to 5-7.
The invention also provides a using method of the universal free nucleic acid protective agent, which comprises the following steps:
after a body fluid sample is extracted by using a sample storage tube containing the universal free nucleic acid protective agent, the sample storage tube is immediately inverted for 5 times, the wrist is completely rotated for 180 degrees during inversion, and the contents of the storage tube are not mixed after the inversion is finished.
The volume ratio of the universal free nucleic acid protective agent to the body fluid sample is 1: 20-100.
The complete rotation of the wrist by 180 degrees is as follows: the palm turns inward to the back.
The invention also provides application of the universal free nucleic acid protective agent in subsequent molecular biology.
The protective agent can be used for subsequent molecular biology by combining free nucleic acid obtained by a free nucleic acid purification kit.
The subsequent molecular biology includes PCR, qPCR, rt-qPCR, methylation sensitive PCR, Southern blot analysis, gene expression analysis, microarray and NGS.
The universal free nucleic acid protective agent is liquid, exists in a nuclease-free plastic tube, is stored at normal temperature, and can protect the degradation of free nucleic acid and the release of background nucleic acid.
Advantageous effects
(1) The invention relates to a total free nucleic acid protective agent, which can protect free DNA and free RNA and prevent the breakage of nucleated cells.
(2) The invention is applicable to liquid body samples, including blood, urine, alveolar lavage fluid, cerebrospinal fluid and other body fluids.
(3) The sample preserved by the method can be preserved for 28 days at normal temperature.
(4) The samples preserved by the method can be transported at ambient temperature.
Drawings
FIG. 1 shows the cfDNA detection results after whole nucleic acid extraction is performed for 0-28 days by using a universal free nucleic acid protective agent, a Norgen cf-DNA/cf-RNA storage tube and a conventional EDTA blood collection tube in the embodiment 1 of the invention to store blood samples.
FIG. 2 shows the cfRNA detection results after whole nucleic acid extraction is performed for 0-28 days by using a universal free nucleic acid protective agent and a Norgen cf-DNA/cf-RNA preservation tube to preserve a blood sample in the embodiment 1 of the invention.
FIG. 3 shows the cfDNA detection results after total nucleic acid extraction is performed for 0-28 days by using a universal free nucleic acid protective agent, a Norgen cf-DNA/cf-RNA preservation tube and a conventional EDTA blood collection tube to preserve urine samples in example 2 of the invention.
FIG. 4 shows the cfRNA detection results after whole nucleic acid extraction is performed for 0-28 days by using a universal free nucleic acid protective agent and a Norgen cf-DNA/cf-RNA preservation tube to preserve a urine sample in the preservation tube of the preservation tube in example 2 of the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
In the embodiment of the invention, 1mL of universal free nucleic acid protective agent comprises the following components: 10mM EDTA, 10mM Tris-HCl, 0.02M dithiothreitol, 5mg glycine, 5mg glutamine, 5mg cysteine, 0.2M trehalose, 0.1mg sodium fluoride, 0.2mg sodium hydroxymethylglycinate, 0.02mM diethyl pyrocarbonate, adjusted to pH 7.
Example 1
For a normal human whole blood sample, the general free nucleic acid protective agent is used for carrying out a comparative experiment with a conventional EDTA blood collection tube and a Norgen cf-DNA/cf-RNA preservation tube.
Taking 6 normal human whole blood samples, dividing the samples into three groups, wherein the group 1 is a conventional EDTA blood collection tube and collects 2ml of blood and shakes uniformly; group 2 is that 2ml blood collected by a Norgen cf-DNA/cf-RNA storage tube is shaken up; group 3 is a storage tube containing the universal free nucleic acid protectant (200. mu.L) of the present invention, 2ml of blood was collected and shaken up. The shaking method is that the sample storage tube is inverted 5 times immediately after the blood is added to ensure that the additives in the tube are uniformly contacted with the sample, the wrist is completely rotated 180 degrees when the tube is inverted, and the palm is turned inwards to the back of the hand. After this step is complete, the contents of the storage tube are not mixed again. The blood storage tube was left for 0 day, 7 days, 14 days, 21 days, and 28 days, and then Nucleic Acid was extracted, and 1ml of a sample was taken with a QIAamp Circulating Nucleic Acid Kit (Kajie Biotechnology, Inc., cat # 55114) to extract all Nucleic acids. And (3) after obtaining the whole free nucleic acid sample, respectively detecting the cfDNA and the cfRNA.
Detection of cfDNA: the obtained total free nucleic acid is subjected to fluorescent quantitative PCR verification to detect the beta-actin gene of the internal reference gene, the result is shown in figure 1, the concentration of the added nucleic acid sample is detected through the size reaction of the Ct value, and the size of the Ct value is inversely proportional to the concentration of the nucleic acid sample. The result shows that the Ct value of the detection of the beta-actin gene in the conventional EDTA blood collection tube is obviously reduced along with the time, which indicates that the nucleated cells are broken, the genome DNA is released into the sample tube, and the conventional EDTA blood collection tube is not suitable for storing the free DNA; although the Ct value of the sample stored by the universal free nucleic acid protective agent and the Norgen cf-DNA/cf-RNA storage tube tends to become smaller with the passage of time through detection, the sample does not have obvious fluctuation change, and the universal free nucleic acid protective agent and the sample stored by the Norgen cf-DNA/cf-RNA storage tube have equivalent effects on the cfDNA and can protect the sample for 28 days.
Detection of cfRNA: and (3) carrying out fluorescent quantitative PCR verification on the obtained total free nucleic acid, detecting miRNA miR-21, carrying out reverse transcription, and then carrying out fluorescent quantitative PCR detection, wherein the result is shown in figure 2, the concentration of the added nucleic acid sample is obtained through the size reaction for detecting the Ct value, and the Ct value is inversely proportional to the concentration of the nucleic acid sample. The result shows that the sample preserved by the universal free nucleic acid protective agent and the Norgen cf-DNA/cf-RNA preservation tube has a tendency that the Ct value becomes smaller with the passage of time, but does not have obvious fluctuation change.
Example 2
For normal human urine samples, the general free nucleic acid protective agent disclosed by the invention is used for carrying out comparison experiments with a conventional EDTA blood collection tube and a Norgen cf-DNA/cf-RNA preservation tube.
Taking 6 normal human urine samples, dividing the urine samples into three groups, wherein the group 1 is a conventional urine collecting pipe and collects 2ml of urine and shakes uniformly; group 2 is that a Norgen cf-DNA/cf-RNA storage tube collects 2ml of urine liquid and shakes evenly; group 3 is a storage tube containing the universal free nucleic acid protectant (200. mu.L) of the present invention, 2ml of urine was collected and shaken up. The shaking method is that the sample storage tube is inverted 5 times immediately after the urine is added to ensure that the additive in the tube is uniformly contacted with the sample, the wrist is completely rotated 180 degrees when the sample storage tube is inverted, and the palm is turned inwards to the back of the hand. After this step is complete, the contents of the storage tube are not mixed again. The blood storage tube was left for 0 day, 7 days, 14 days, 21 days, and 28 days, and then Nucleic Acid was extracted, and 1ml of a sample was taken with a QIAamp Circulating Nucleic Acid Kit (Kajie Biotechnology, Inc., cat # 55114) to extract all Nucleic acids. And (3) after obtaining the whole free nucleic acid sample, respectively detecting the cfDNA and the cfRNA.
Detection of cfDNA: the obtained total free nucleic acid is subjected to fluorescent quantitative PCR verification, and the beta-actin gene of the internal reference gene is detected, and the result is shown in figure 3, the concentration of the added nucleic acid sample is detected through the size reaction of the Ct value, and the size of the Ct value is inversely proportional to the concentration of the nucleic acid sample. The result shows that the Ct value of the detection of the beta-actin gene in the conventional EDTA blood collection tube is obviously reduced along with the time, which indicates that the nucleated cells are broken, the genome DNA is released into the sample tube, and the conventional EDTA blood collection tube is not suitable for storing the free DNA; although the Ct value of the sample stored by the universal free nucleic acid protective agent and the Norgen cf-DNA/cf-RNA storage tube tends to become smaller with the passage of time through detection, the sample does not have obvious fluctuation change, and the universal free nucleic acid protective agent and the sample stored by the Norgen cf-DNA/cf-RNA storage tube have equivalent effects on the cfDNA and can protect the sample for 28 days.
Detection of cfRNA: and (3) carrying out fluorescent quantitative PCR verification on the obtained total free nucleic acid, detecting miRNA miR-21, carrying out reverse transcription, and then carrying out fluorescent quantitative PCR detection, wherein the result is shown in figure 4, the concentration of the added nucleic acid sample is obtained through the size reaction for detecting the Ct value, and the Ct value is inversely proportional to the concentration of the nucleic acid sample. The result shows that the sample preserved by the universal free nucleic acid protective agent and the Norgen cf-DNA/cf-RNA preservation tube has a tendency that the Ct value becomes smaller with the passage of time, but does not have obvious fluctuation change.
Claims (4)
1. A universal free nucleic acid protectant, wherein the protectant component comprises: 2-200mM of ethylenediamine tetraacetic acid, 5-20mM of Tris-HCl, 0.02-2M of dithiothreitol, 0.1-30mg/mL of glycine, 0.1-30mg/mL of glutamine, 0.1-20mg/mL of cysteine, 0.2-1M of trehalose, 0.1-20mg/mL of sodium fluoride, 0.2-30mg/mL of sodium hydroxymethyl glycinate and 0.02-0.5mM of diethyl pyrocarbonate, and adjusting the pH value to 5-7.
2. Use of a protective agent according to claim 1 in molecular biology.
3. Use according to claim 2, wherein the protective agent is bound to free nucleic acid obtained from a free nucleic acid purification kit and is used in subsequent molecular biology.
4. Use according to claim 2 or 3, wherein the subsequent molecular biology comprises PCR, qPCR, rt-qPCR, methylation sensitive PCR, Southern blot analysis, gene expression analysis, microarray and NGS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011414505.4A CN112501159A (en) | 2020-12-04 | 2020-12-04 | Universal free nucleic acid protective agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011414505.4A CN112501159A (en) | 2020-12-04 | 2020-12-04 | Universal free nucleic acid protective agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112501159A true CN112501159A (en) | 2021-03-16 |
Family
ID=74970724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011414505.4A Pending CN112501159A (en) | 2020-12-04 | 2020-12-04 | Universal free nucleic acid protective agent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112501159A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110111410A1 (en) * | 2009-11-09 | 2011-05-12 | Streck, Inc. | Stabilization of rna in intact cells within a blood sample |
| US20130034860A1 (en) * | 2009-02-18 | 2013-02-07 | Streck, Inc. | Preservation of cell-free rna in blood samples |
| CN106900692A (en) * | 2017-03-06 | 2017-06-30 | 上海迅伯生物科技有限公司 | A kind of preservative agent composition of blood DNA |
| CN108893524A (en) * | 2018-06-04 | 2018-11-27 | 北京启衡星生物科技有限公司 | The protective agent of dissociative DNA in blood plasma |
-
2020
- 2020-12-04 CN CN202011414505.4A patent/CN112501159A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130034860A1 (en) * | 2009-02-18 | 2013-02-07 | Streck, Inc. | Preservation of cell-free rna in blood samples |
| US20110111410A1 (en) * | 2009-11-09 | 2011-05-12 | Streck, Inc. | Stabilization of rna in intact cells within a blood sample |
| CN106900692A (en) * | 2017-03-06 | 2017-06-30 | 上海迅伯生物科技有限公司 | A kind of preservative agent composition of blood DNA |
| CN108893524A (en) * | 2018-06-04 | 2018-11-27 | 北京启衡星生物科技有限公司 | The protective agent of dissociative DNA in blood plasma |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP4219743A1 (en) | Sample nucleic acid measurement test kit, reagent, and application thereof | |
| CN110982876B (en) | Pretreatment method, pretreatment liquid, kit and application of virus nucleic acid detection | |
| CN103725796B (en) | Mark parting fluorescence PCR detection kit and preparation thereof in a kind of bovine viral diarrhea virus | |
| CN112831604A (en) | Pathogenic microorganism detection primer group, kit and method based on targeted sequencing | |
| JP2019521640A (en) | Protein-based sample collection matrix and apparatus | |
| US20160017315A1 (en) | Methods for one step nucleic acid amplification of non-eluted samples | |
| CN103757139A (en) | Canine distemper virus and canine parvovirus duplex TaqMan-MGB fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof | |
| Marsh et al. | A laboratory methodology for dual RNA-sequencing of bacteria and their host cells in vitro | |
| Farkas et al. | Specimen collection and storage for diagnostic molecular pathology investigation | |
| Yu et al. | Comparative evaluation of three preprocessing methods for extraction and detection of influenza A virus nucleic acids from sputum | |
| TWI362419B (en) | Nucleic acid detection | |
| CN110819625A (en) | Method for extracting genome DNA (deoxyribonucleic acid) suitable for bacteria and/or fungi | |
| CN116891849B (en) | Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method | |
| US20220025362A1 (en) | DNA stabilization of RNA | |
| US11434527B2 (en) | Method for detecting mycoplasma using mitochondrial DNA as internal control sample | |
| CN112501159A (en) | Universal free nucleic acid protective agent | |
| WO2017198715A1 (en) | Nucleic acid stabilization agent | |
| US10160965B2 (en) | Method and materials for nucleic acids extraction and purification | |
| CN101845515B (en) | Method for detection of swine influenza A H1N1 virus based on pyrosequencing technology | |
| Biswal et al. | Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis | |
| CN106834538A (en) | One kind differentiates Porcine epidemic diarrhea virus vaccine low virulent strain and/or popular strain one-step method RT PCR diagnostic kits | |
| CN112063615A (en) | Genome DNA extracting solution, genome DNA extracting method and application thereof | |
| KR20220047117A (en) | Reagent composition including sodium dodecyl sulfate for extracting nucleic acid from plant sample and nucleic acid extraction kit for plant sample | |
| CN110804611A (en) | Method for extracting genome DNA (deoxyribonucleic acid) suitable for bacteria and/or fungi | |
| CN104862418A (en) | Specific primers for detecting European-type porcine reproductive and respiratory syndrome viruses and corresponding detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210316 |
|
| RJ01 | Rejection of invention patent application after publication |