CN111172240A - Nucleic acid releasing agent and HPV (human papilloma Virus) nucleic acid detection kit - Google Patents
Nucleic acid releasing agent and HPV (human papilloma Virus) nucleic acid detection kit Download PDFInfo
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Abstract
The invention discloses a nucleic acid releasing agent, which comprises the following components in percentage by mass: (1-3) Triton X-100 and diethylene glycol monoethyl ether (DCDE). The low-price composition of Triton X-100 and DCDE is adopted to replace the traditional guanidine salt and detergent, and the obtained nucleic acid releasing agent has the advantages of safe use, easy degradation, simple and convenient operation and low manufacturing cost, can effectively crack a serum sample containing pathogens, quickly release the nucleic acid in the serum sample, reduce the inhibition to downstream experiments, and has high pathogen cracking efficiency. The invention also discloses an HPV virus nucleic acid detection kit which comprises the nucleic acid releasing agent, is simple and convenient to operate, has high working efficiency, effectively avoids the link of possible pollution in the experimental operation process, reduces the loss of HPV DNA in the extraction process, and has the quantitative limit of 45IU/mL and the detection limit of 22 IU/mL.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nucleic acid releasing agent and an HPV nucleic acid detection kit.
Background
The real-time fluorescent quantitative PCR detection of free virus in blood is the most advanced detection method in clinical at present, and has been developed to be quite mature at present, but the steps of extracting virus nucleic acid before detection are quite complicated, such as: (1) the traditional boiling method comprises the following steps: directly mixing the alkaline lysate with the pathogenic liquid, performing high-temperature lysis, and centrifuging to obtain nucleic acid; (2) concentration and boiling method: firstly, polysaccharide is used for concentrating and precipitating pathogen, then alkaline lysate is added for high-temperature cracking, and nucleic acid is obtained through centrifugation; (3) centrifugal column method: adsorbing, purifying and eluting by a silica gel membrane or a glass fiber membrane to obtain nucleic acid; (4) magnetic bead method: and adsorbing, purifying and eluting by using silica powder or polyvinylidene coated magnetic beads to obtain the nucleic acid. The method needs to be subjected to cracking, repeated washing, elution and the like, is time-consuming and labor-consuming, is very easy to pollute, can generate more wastes, and is not beneficial to environmental protection. Nucleic acid extraction is the starting point for downstream nucleic acid detection, research or product development, and the quality, purity and integrity of the isolated nucleic acid directly affects the research or diagnostic results, so nucleic acid releasing agents play a very important role in the field of virus detection.
Nucleic acids obtained from the ideal nucleic acid releasing agent are free from contamination with proteins, lipids or other nucleic acids, and many specialized nucleic acid releasing agents are available to obtain the desired nucleic acid from biological samples. For example, chinese patent 201310009055.4 discloses a one-step method for fluorescence quantitative PCR detection of pathogen nucleic acid, which comprises selecting a conserved gene sequence for the pathogen nucleic acid to be detected, designing a specific primer probe, extracting the pathogen nucleic acid with a nucleic acid quick release reagent, and directly adding the corresponding PCR reaction solution to realize the fluorescence quantitative detection of PCR.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a nucleic acid releasing agent, which adopts a mixture of low-cost Triton X-100 and diethylene glycol monoethyl ether (DCDE) to replace the traditional detergent, so that the nucleic acid releasing agent has the advantages of safe use, easy degradation, simple and convenient operation and low manufacturing cost, can effectively crack a serum sample containing pathogens, fully release the nucleic acid in the serum sample, reduce the inhibition to downstream experiments, has high pathogen cracking efficiency, and can shorten the extraction time to 5-10 minutes.
The invention also aims to provide an HPV virus nucleic acid detection kit which comprises the nucleic acid releasing agent, is simple and convenient to operate and high in working efficiency, effectively avoids the link of possible pollution in the experimental operation process, reduces the loss of pathogen DNA in the extraction process, and has the limit of quantification of 45IU/mL and the limit of detection of 22 IU/mL.
One of the purposes of the invention is realized by adopting the following technical scheme:
a nucleic acid releasing agent comprises a combination of Triton X-100 and diethylene glycol monoethyl ether; the mass ratio of the Triton X-100 to the diethylene glycol monoethyl ether is 1: (1-3).
Further, the nucleic acid releasing agent also comprises sodium hydroxide, ethanol, sodium dodecyl sulfate and sterile water.
Further, the nucleic acid releasing agent comprises 5-20 wt.% of a combination of Triton X-100 and diethylene glycol monoethyl ether, 50-300mmol/L of sodium hydroxide, 0.2-1 Vol.% of ethanol, 0.01-1 wt.% of sodium dodecyl sulfate, and the balance of sterile water.
Preferably, the nucleic acid releasing agent comprises 10 wt.% of a combination of triton x-100 and diethylene glycol monoethyl ether, 150mmol/L sodium hydroxide, 0.8 Vol.% ethanol, 0.5 wt.% sodium dodecyl sulfate, and the balance sterile water.
Further, after the nucleic acid releasing agent and a sample to be detected containing a pathogen are uniformly mixed, standing for 5-10min at room temperature to obtain the target nucleic acid.
The volume ratio of the nucleic acid releasing agent to the sample to be detected is 1: (1-3).
Preferably, the volume ratio of the nucleic acid releasing agent to the sample to be tested is 1: 2.
the sample to be detected is a serum sample, a plasma sample or a genital secretion sample.
The second purpose of the invention can be achieved by adopting the following technical scheme:
an HPV virus nucleic acid detection kit comprises the nucleic acid releasing agent, PCR reaction liquid and an internal standard.
Preferably, the volume ratio of the nucleic acid releasing agent to the PCR reaction solution to the internal standard is 5:40: 0.2.
Compared with the prior art, the invention has the beneficial effects that:
1. the surfactant (such as sarsantine and the like) adopted by the existing nucleic acid release agent is expensive (such as the price of 10mg sarsantine is 2815 yuan), so that the experiment cost is greatly increased; meanwhile, impurities are inevitably introduced to influence the subsequent experiment. The invention adopts the composition of low-cost Triton X-100 and diethylene glycol monoethyl ether (DCDE) as the core component of the nucleic acid releasing agent, compared with the traditional quaternary ammonium salt cationic surfactant, the composition has higher capability and efficiency of reducing surface tension, and the mixed surfactant of Triton X-100 and DCDE can also enhance the function among all components and play a role in quick cracking; and Triton X-100 and DCDE are chemical synthesis products, and have clear components, are easy to degrade and cannot influence subsequent experiments.
2. The micro nucleic acid releasing agent can destroy the coat protein structure of pathogen fast, and make the matter of serum protein and other interference PCR amplification closed and precipitate and make the DNA nucleic acid of HPV virus to be separated out fully. According to the invention, Triton X-100 and DCDE are used to replace the traditional guanidine salt and detergent, so that the nucleic acid releasing agent has the advantages of safe use, simple and convenient operation and low manufacturing cost, can effectively crack a serum sample containing pathogens, release nucleic acid in the serum sample and reduce the inhibition to downstream experiments, and pathogen DNA is fully released without interfering the amplification of subsequent PCR, thereby remarkably improving the cracking efficiency of the pathogens and shortening the extraction time to 5-10 minutes.
3. The kit does not need complicated extraction steps such as cracking, washing, elution and the like the traditional nucleic acid extraction kit, greatly improves the working efficiency of a laboratory, effectively avoids the link of possible pollution in the experimental operation process, and simultaneously reduces the generation of medical wastes. The method reduces the loss of pathogen DNA in the extraction process, and the quantitative limit of the corresponding kit for detecting HPV nucleic acid is 45IU/mL, and the detection limit is 22 IU/mL.
4. The components of the nucleic acid releasing agent are low in toxicity and easy to degrade, the stability of the nucleic acid releasing agent is better, and the nucleic acid releasing agent can still keep very good stability within 6 months under the environments of low temperature, normal temperature and high temperature.
Drawings
FIG. 1 is a PCR test chart of serum standards with HPV DNA titers of 25IU/mL, 250IU/mL, 2500IU/mL, 25000IU/mL, 250000 IU/mL; in the figure: 1. 250000 IU/mL; 2. 25000 IU/mL; 3. 2500 IU/mL; 4. 250/mL; 5. 25 IU/mL;
FIG. 2 is a PCR assay of a serum standard with an HPV DNA titer of 22 IU/mL; in the figure: 1. internal standard; 2. 22 IU/mL;
FIG. 3 is a PCR assay of a serum standard with an HPV DNA titer of 45 IU/mL; in the figure: 1. internal standard; 2. 45 IU/mL;
FIG. 4 is a specific PCR assay of HBV, HSV, CT, NG DNA in vivo; in the figure: 1. internal standard; 2. the pathogens HBV, HSV, CT and NG.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
A nucleic acid releasing agent comprises a combination of Triton X-100 and diethylene glycol monoethyl ether, sodium hydroxide, ethanol, sodium dodecyl sulfate and sterile water; wherein the mass ratio of Triton X-100 to diethylene glycol monoethyl ether is 1: (1-3).
By using the combined surfactant of Triton X-100 and diethylene glycol monoethyl ether instead of the traditional guanidine salt and a part of detergent, the serum sample containing virus particles can be effectively cracked, the nucleotide in the serum sample can be released, and the inhibition of downstream experiments can be reduced, so that the virus cracking efficiency can be obviously improved, and the detection time can be greatly shortened.
As a further preferred embodiment, the nucleic acid releasing agent comprises 5-20 wt.% of a combination of Triton X-100 and diethylene glycol monoethyl ether, 50-300mmol/L sodium hydroxide, 0.2-1 Vol.% ethanol, 0.01-1 wt.% sodium dodecyl sulfate, the balance being sterile water.
As a further preferred embodiment, the nucleic acid releasing agent comprises 10 wt.% of a combination of Triton X-100 and diethylene glycol monoethyl ether, 150mmol/L sodium hydroxide, 0.8 Vol.% ethanol, 0.5 wt.% sodium dodecyl sulfate, the balance being sterile water.
Mixing the nucleic acid releasing agent and a sample to be detected containing pathogens, inserting the mixture into the bottom of the mixed solution by using a pipette gun for sucking and beating for multiple times, and standing for 5-10min at room temperature to obtain the target nucleic acid.
As a further preferable scheme, the volume ratio of the nucleic acid releasing agent to the sample to be tested is 1: (1-3). The sample to be detected is a serum sample, a plasma sample or a genital secretion sample.
An HPV virus nucleic acid detection kit comprises the nucleic acid releasing agent, PCR reaction liquid and an internal standard, wherein the volume ratio of the nucleic acid releasing agent to the PCR reaction liquid to the internal standard is 5:40: 0.2.
Example 1
A nucleic acid releasing agent comprises 5 wt.% of Triton X-100 and DCDE composition, 50mmol/L NaOH, 0.2 Vol.% ethanol, 1 wt.% sodium dodecylsulfate, and the balance water. Wherein the mass ratio of Tritonx-100 to DCDE is 1: 3.
Example 2
A nucleic acid releasing agent comprises 10 wt.% of Triton X-100 and DCDE composition, 100mmol/L sodium hydroxide, 0.8 Vol.% ethanol, 0.5 wt.% sodium dodecyl sulfate, and water in balance. Wherein the mass ratio of Tritonx-100 to DCDE is 1: 2.
Example 3
A nucleic acid releasing agent comprises 20 wt.% of a combination of Triton X-100 and DCDE, 200mmol/L sodium hydroxide, 1 Vol.% ethanol, 0.8 wt.% sodium dodecyl sulfate, and the balance water. Wherein the mass ratio of Tritonx-100 to DCDE is 1: 1.
Example 4
An HPV virus nucleic acid detection kit comprises a nucleic acid releasing agent, PCR reaction liquid and an internal standard, wherein:
the internal standard is:
CATTAGAACAGCAATACAACAAACTGTTGTGTGATTTGTTAATTAGGTGTAT TAACTGTCAAAAGCCACTGTGTCCTGAAGAAAAGCAAAGACATCTGGACA AAAAGCAAAGATTCCATAACATGAGGGGTCGGTGGACCGGTCGATGTATG TCTTGTTG
the PCR reaction solution comprises: primer, probe, 15-25mM Tris-HCl pH 8.0, 15-25mM KCl, 2.5-5mM (NH)4)2SO4，2-5mM MgCl20.5-2mM dNTP/UTP Mix. Wherein, the primers comprise 0.2 mu mol/L internal standard primer and 0.3 mu mol/LHPV detection primer, and the probes comprise 0.3 mu mol/L internal standard probe and 0.3 mu mol/LHPV detection probe, as follows:
an internal standard primer: AGCCACTGTGTCCTGAAGAA/TCCACCGACCCCTCATGTTA internal standard probe: GCAAAGACATCTGGACAA
HPV detection primers:
ATAGAGGCCAGTGCCATTCG/TGGAGTCTTTCCTGTCGTGC
HPV detection probe: GGAAAGACTCCAAT
The HPV nucleic acid detection kit also comprises a strong positive control substance, a critical positive control substance, a negative control substance and a positive quantitative reference substance.
Example 5
A method for detecting HPV nucleic acid in serum, which comprises the following steps:
1) the components in the HPV nucleic acid detection kit in example 4 were returned to room temperature, shaken and centrifuged instantaneously.
2) And (3) taking 40 mu l of PCR reaction solution and 0.2 mu l of internal standard, fully and uniformly mixing to obtain PCR-mix, and performing instantaneous centrifugation for later use.
3) Taking a PCR reaction tube, adding 5ul of nucleic acid releasing agent into each tube, adding 10ul of serum to be detected, inserting the tube into the bottom of the PCR reaction tube by using a pipette gun, sucking and beating for 4 times to mix uniformly, standing at room temperature for 4 minutes, adding 40.2ul of PCR-mix obtained in the step 2), performing instantaneous centrifugation, and then placing the tube in an ABI7500/Roche Light Cycler 450 real-time fluorescence quantitative PCR instrument for amplification.
The PCR amplification conditions were: 6 minutes at 37 ℃; 5 minutes at 95 ℃; 95 ℃, 10 seconds, 60 ℃, 30 seconds, 45 cycles.
Example 6
In this example, the nucleic acid releasing agent of example 2 and the test kit of example 4 were used to test serum standards with HPV DNA titers of 25IU/mL, 250/mL, 2500IU/mL, 25000IU/mL, and 250000IU/mL concentration gradients according to the test method of example 5, and the test results are shown in FIG. 1.
Example 7
In this example, the nucleic acid releasing agent of example 2 and the detection kit of example 4 were used to detect a serum standard substance having an HPV DNA titer of 22IU/mL according to the detection method of example 5, and the detection results are shown in FIG. 2. As can be seen from FIG. 2, the kit of the present invention can detect HPV DNA of 22 IU/mL.
Example 8
In this example, the nucleic acid releasing agent of example 2 and the detection kit of example 4 were used to detect a serum standard substance having an HPV DNA titer of 45IU/mL according to the detection method of example 5, and the detection results are shown in FIG. 3. As can be seen from FIG. 3, the kit of the present invention can quantitatively detect HPV DNA of 45 IU/mL.
Example 9
In this embodiment, the nucleic acid releasing agent of embodiment 2 and the detection kit of embodiment 4 are used to detect serum samples containing DNA of pathogens HBV, HSV, CT, NG, respectively, according to the detection method of embodiment 5, and the detection results are shown in fig. 4, and as can be seen from fig. 4, only an internal standard amplification curve is obtained, and no amplification curve occurs in the pathogens, which indicates that the kit of the present invention has good specificity.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of the present invention claimed in the present invention.
Claims (10)
1. A nucleic acid releasing agent comprising a combination of Triton X-100 and diethylene glycol monoethyl ether; the mass ratio of the Triton X-100 to the diethylene glycol monoethyl ether is 1: (1-3).
2. The nucleic acid releasing agent of claim 1 further comprising sodium hydroxide, ethanol, sodium dodecyl sulfate and sterile water.
3. The nucleic acid releasing agent according to claim 2, wherein the nucleic acid releasing agent comprises 5 to 20 wt.% of a combination of Triton X-100 and diethylene glycol monoethyl ether, 50 to 300mmol/L of sodium hydroxide, 0.2 to 1 Vol.% of ethanol, 0.01 to 1 wt.% of sodium dodecyl sulfate, and the balance being sterile water.
4. The nucleic acid releasing agent according to claim 3, wherein the nucleic acid releasing agent comprises 10 wt.% of a combination of Triton X-100 and diethylene glycol monoethyl ether, 150mmol/L sodium hydroxide, 0.8 Vol.% ethanol, 0.5 wt.% sodium dodecyl sulfate, and the balance being sterile water.
5. The nucleic acid releasing agent according to any of claims 1 to 4, wherein the target nucleic acid is obtained by uniformly mixing the nucleic acid releasing agent with a sample to be tested containing a pathogen and then allowing the mixture to stand at room temperature for 5 to 10 min.
6. The nucleic acid releasing agent according to claim 5, wherein the volume ratio of the nucleic acid releasing agent to the sample to be tested is 1: (1-3).
7. The nucleic acid releasing agent according to claim 5, wherein the volume ratio of the nucleic acid releasing agent to the sample to be tested is 1: 2.
8. the nucleic acid releasing agent according to claim 5, wherein the sample to be tested is a serum sample, a plasma sample or a genital secretion sample.
9. An HPV virus nucleic acid detection kit, characterized by comprising a nucleic acid releasing agent, a PCR reaction solution and an internal standard, wherein the nucleic acid releasing agent is the nucleic acid releasing agent of any one of claims 1 to 4.
10. The HPV viral nucleic acid detection kit of claim 9, wherein the nucleic acid releasing agent, the PCR reaction solution, and the internal standard are in a volume ratio of 5:40: 0.2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808847A (en) * | 2020-08-12 | 2020-10-23 | 济南国益生物科技有限公司 | Release agent for rapidly extracting nucleic acid by one-step method and preparation and use methods thereof |
| CN113080187A (en) * | 2021-03-31 | 2021-07-09 | 上海纽脉医疗科技有限公司 | Composition for removing phospholipids and cell debris and method for removing phospholipids and cell debris from biological tissue |
| CN113186037A (en) * | 2021-04-23 | 2021-07-30 | 山东博弘基因科技有限公司 | Nucleic acid scavenger |
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| WO2007050327A2 (en) * | 2005-10-21 | 2007-05-03 | Qiagen North American Holdings, Inc. | Compact apparatus, compositions and methods for purifying nucleic acids |
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| CN101124321A (en) * | 2004-11-05 | 2008-02-13 | 恰根北美控股有限公司 | Compositions and methods for purifying nucleic acids from stabilization reagents |
| WO2007050327A2 (en) * | 2005-10-21 | 2007-05-03 | Qiagen North American Holdings, Inc. | Compact apparatus, compositions and methods for purifying nucleic acids |
Non-Patent Citations (1)
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808847A (en) * | 2020-08-12 | 2020-10-23 | 济南国益生物科技有限公司 | Release agent for rapidly extracting nucleic acid by one-step method and preparation and use methods thereof |
| CN113080187A (en) * | 2021-03-31 | 2021-07-09 | 上海纽脉医疗科技有限公司 | Composition for removing phospholipids and cell debris and method for removing phospholipids and cell debris from biological tissue |
| CN113080187B (en) * | 2021-03-31 | 2022-06-21 | 上海纽脉医疗科技有限公司 | Composition for removing phospholipids and cell debris and method for removing phospholipids and cell debris from biological tissue |
| WO2022206425A1 (en) * | 2021-03-31 | 2022-10-06 | 上海纽脉医疗科技股份有限公司 | Composition for removing phospholipids and cell debris and method for removing phospholipids and cell debris on biological tissue |
| CN113186037A (en) * | 2021-04-23 | 2021-07-30 | 山东博弘基因科技有限公司 | Nucleic acid scavenger |
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