CN113621683A - Application of phase-change paraffin as reagent separation structure of column type detection card - Google Patents
Application of phase-change paraffin as reagent separation structure of column type detection card Download PDFInfo
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- CN113621683A CN113621683A CN202110313070.2A CN202110313070A CN113621683A CN 113621683 A CN113621683 A CN 113621683A CN 202110313070 A CN202110313070 A CN 202110313070A CN 113621683 A CN113621683 A CN 113621683A
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- 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
Abstract
The invention relates to the field of in-vitro diagnosis nucleic acid detection, and provides application of phase-change paraffin as a reagent separation structure of a column type detection card. The application not only can greatly simplify the structure of the column type detection card, but also can not lose the magnetic beads. And after the nucleic acid amplification reaction is finished, the heated paraffin floats to the upper part of the lysis solution to be solidified again to seal the whole reaction system, so that the leakage of waste liquid and biomass contained in the waste liquid can be avoided.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis nucleic acid detection, in particular to application of a phase-change paraffin as a reagent separation structure of a column type detection card.
Background
The magnetic bead method is an automatic nucleic acid extraction method used for nucleic acid detection. The magnetic bead method is characterized in that the magnetic attraction effect of an external magnet and magnetic beads in a test tube is utilized, the external magnet drives the magnetic beads to move in the test tube, the magnetic bead-nucleic acid complex moves from lysis solution to washing solution and then from the washing solution to amplification solution, and therefore the automatic extraction of a nucleic acid template is achieved.
The test tube containing the amplification solution, the lysis solution and the washing solution is called a column type detection card, wherein a separation structure is arranged between two adjacent layers of liquid, namely between the lysis solution and the washing solution and between the washing solution and the amplification solution, so as to avoid the adjacent liquids from being mixed. The current column type detection card adopts a mechanical separation structure, the mechanical separation structure comprises a plunger, a hydrophobic material (liquid) and a spring, the plunger is positioned between two layers of liquid under the action of the spring in a non-use state, the reliable separation of the two layers of liquid is ensured, when the column type detection card is used, external force is applied to ensure that the plunger overcomes the action of the spring to leave between the two layers of liquid, and the hydrophobic material moves between the two layers of liquid, so that a magnetic bead-nucleic acid complex can be transmitted between the layers of liquid.
The column type detection card has the following technical defects: due to the adoption of the mechanical separation structure, the column type detection card has a complex structure, magnetic beads are easily lost, the amount of the template is reduced, and the detection sensitivity is influenced.
Disclosure of Invention
The present invention is directed to a use of phase-change paraffin as a reagent separation structure of a column-type detection card, so as to solve the above technical defects of the related art.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the application of phase-change paraffin as reagent separating structure of column-type detecting card is disclosed. That is, the existing mechanical partition structure is replaced with phase-change paraffin.
In the application, before the nucleic acid amplification reaction, the phase-change paraffin is in a solid state and is positioned between two layers of liquid, after the nucleic acid amplification reaction is heated, the phase-change paraffin is changed into a liquid state and rises to the surface of the top layer of liquid, and then the phase-change paraffin is solidified to form a seal for the liquid.
In the above application, preferably, the melting point of the phase-change paraffin is within the enzyme activity temperature range of the amplification solution.
Compared with the prior art, the invention has at least the following beneficial effects:
not only can greatly simplify the structure of the column type detection card, but also can not lose the magnetic beads.
After the nucleic acid amplification reaction is finished, the heated paraffin floats to the upper part of the lysis solution to be solidified again to seal the whole reaction system, so that the leakage of waste liquid and biomass contained in the waste liquid can be avoided.
Drawings
FIG. 1 is a diagram showing the effect of paraffin as a partition structure before a nucleic acid amplification reaction;
FIG. 2 is a graph showing the effect of paraffin as a partition structure after a nucleic acid amplification reaction;
FIG. 3 is a graph showing the effect of separation of a high melting point paraffin as a separation structure before a nucleic acid amplification reaction;
FIG. 4 is a graph showing the effect of a high melting point paraffin as a partition structure after a nucleic acid amplification reaction;
FIG. 5 is a nucleic acid amplification curve using a high melting point paraffin as a partition structure;
FIG. 6 is a nucleic acid amplification curve using low melting point paraffin as a partition structure.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The invention provides a new application of paraffin, namely application of phase-change paraffin as a reagent separation structure of a column type detection card.
In the application, before the nucleic acid amplification reaction, the phase-change paraffin is in a solid state and is positioned between two layers of liquid, after the nucleic acid amplification reaction is heated, the phase-change paraffin is changed into a liquid state and rises to the surface of the liquid at the top layer, and then the phase-change paraffin is solidified to form a seal for the liquid.
This application can make the column type detect the card on the one hand and save mechanical type partition structure, and the structure can be simplified greatly, can effectively avoid the magnetic bead to be blocked at mechanical type partition structure or block in the joint portion of mechanical type partition structure and test tube moreover, and then can avoid losing the template volume that the magnetic bead caused and reduce the technical defect that detection sensitivity reduces. On the other hand, after the reaction is finished, the heated paraffin floats to the upper part of the cracking liquid to be solidified again to seal the whole reaction system, so that the leakage of waste liquid and the biomass contained in the waste liquid can be avoided.
The melting point of the phase-change paraffin has an influence on the separation effect and the sealing effect. The low-melting-point paraffin has an unstable solidification structure, cannot be stored for a short time at normal temperature, has a high melting temperature and is long in time, and the enzyme activity is influenced. In the present application, the melting point of the phase-change paraffin is preferably within the enzyme activity temperature range of the amplification solution. For example, paraffin with a melting point of 45-70 ℃ can be selected for the conventional temperature-variable nucleic acid amplification reaction, and paraffin with a melting point of 5-50 ℃ can be selected for the normal-temperature or low-temperature isothermal nucleic acid amplification reaction.
Separation effect and sealing effect experiment:
paraffin wax separation and sealing experiment
Pre-nucleic acid amplification reaction state: two layers of liquid are separated by an upper layer of paraffin and a lower layer of paraffin, and after the liquid is horizontally vibrated for three days at the rotating speed of 180rpm, as shown in figure 1, the white substance in the figure 1 is the paraffin, the volumes of the two layers of liquid are not obviously changed, and the paraffin is firmly adsorbed with the pipe wall without liquid infiltration.
Post nucleic acid amplification reaction state: as shown in figure 2, after heating, the paraffin on the lower layer melts and floats to the upper layer and is fused with the paraffin on the upper layer to solidify and seal the whole pipe orifice, after horizontal oscillation for three days at the rotating speed of 180rmp, no obvious change occurs, and liquid in the pipe does not seep out.
Therefore, the paraffin can not only meet the reliable separation of liquid before nucleic acid amplification reaction, but also automatically float to the top layer after reaction for solidification, reliably seal the whole reaction system and avoid the leakage of waste liquid and biomass contained in the reaction system.
Second, high melting point paraffin separation and sealing experiment
Pre-nucleic acid amplification reaction state: two layers of liquid are separated by an upper layer of paraffin and a lower layer of paraffin, and after the liquid is horizontally vibrated for three days at the rotating speed of 180rpm, as shown in figure 3, the white substance in figure 3 is the paraffin, the volumes of the two layers of liquid are not obviously changed, and the paraffin is firmly adsorbed with the pipe wall without liquid infiltration.
Post nucleic acid amplification reaction state: as shown in fig. 4, after heating, the paraffin wax on the upper layer melts and then is solidified again to seal the pipe orifice, part of the paraffin wax on the lower layer moves upwards, and the volume of the liquid on the lower layer does not change obviously.
Therefore, the high melting point paraffin can satisfy the reliable separation of the liquid before the nucleic acid amplification reaction, but the lower paraffin only partially moves upwards after the reaction.
Experiment of nucleic acid amplification effect: the high-melting-point paraffin and the low-melting-point paraffin are respectively used as separation structures of liquid in the column type detection card, the column type detection card is used for carrying out nucleic acid amplification detection, amplification curves are respectively shown in figures 5 and 6, the figure 5 is a nucleic acid amplification curve corresponding to the separation of the high-melting-point paraffin, and the figure 6 is a nucleic acid amplification curve corresponding to the separation of the low-melting-point paraffin, so that the fact that the melting of the high-melting-point paraffin needs longer time and temperature is not beneficial to keeping enzyme activity can be seen, the amplification efficiency is reduced along with the prolonging of time, and the selection of the paraffin with the melting point within the enzyme activity temperature range is beneficial to nucleic acid amplification.
The present invention has been described in detail with reference to the specific embodiments, and the detailed description is only for the purpose of helping those skilled in the art understand the present invention, and is not to be construed as limiting the scope of the present invention. Various modifications, equivalent changes, etc. made by those skilled in the art under the spirit of the present invention shall be included in the protection scope of the present invention.
Claims (3)
1. The application of phase-change paraffin as a reagent separation structure of a column type detection card.
2. The use of claim 1, wherein the phase-change paraffin is in a solid state and is disposed between two layers of liquid prior to the nucleic acid amplification reaction, and wherein upon heating of the nucleic acid amplification reaction, the phase-change paraffin changes to a liquid state and rises to the surface of the top liquid, and then solidifies to form a seal with the liquid.
3. Use according to claim 1, wherein the phase-change paraffin wax has a melting point in the temperature range of the enzymatic activity of the amplification fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110313070.2A CN113621683A (en) | 2021-03-24 | 2021-03-24 | Application of phase-change paraffin as reagent separation structure of column type detection card |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110313070.2A CN113621683A (en) | 2021-03-24 | 2021-03-24 | Application of phase-change paraffin as reagent separation structure of column type detection card |
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| CN202110313070.2A Pending CN113621683A (en) | 2021-03-24 | 2021-03-24 | Application of phase-change paraffin as reagent separation structure of column type detection card |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117210309A (en) * | 2023-11-09 | 2023-12-12 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824486A (en) * | 2009-12-30 | 2010-09-08 | 华东医学生物技术研究所 | Method and device for rapidly detecting nucleic acid |
| US20130190277A1 (en) * | 2012-01-25 | 2013-07-25 | Joel Bain HERRON | Paraffin Wax Embedded Drug Delivery System |
| US20160121332A1 (en) * | 2014-10-31 | 2016-05-05 | Seiko Epson Corporation | Nucleic acid amplification reaction apparatus and nucleic acid detection method |
| CN107020057A (en) * | 2017-03-06 | 2017-08-08 | 上海仁度生物科技有限公司 | Isolate the method for reaction system using the phase change transition of paraffin |
| CN111876468A (en) * | 2020-09-03 | 2020-11-03 | 杭州天微基因科技有限公司 | Full-automatic nucleic acid detection method and test tube |
-
2021
- 2021-03-24 CN CN202110313070.2A patent/CN113621683A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824486A (en) * | 2009-12-30 | 2010-09-08 | 华东医学生物技术研究所 | Method and device for rapidly detecting nucleic acid |
| US20130190277A1 (en) * | 2012-01-25 | 2013-07-25 | Joel Bain HERRON | Paraffin Wax Embedded Drug Delivery System |
| US20160121332A1 (en) * | 2014-10-31 | 2016-05-05 | Seiko Epson Corporation | Nucleic acid amplification reaction apparatus and nucleic acid detection method |
| CN107020057A (en) * | 2017-03-06 | 2017-08-08 | 上海仁度生物科技有限公司 | Isolate the method for reaction system using the phase change transition of paraffin |
| CN111876468A (en) * | 2020-09-03 | 2020-11-03 | 杭州天微基因科技有限公司 | Full-automatic nucleic acid detection method and test tube |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117210309A (en) * | 2023-11-09 | 2023-12-12 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
| CN117210309B (en) * | 2023-11-09 | 2024-01-26 | 迈克生物股份有限公司 | Biological sample processing device and biological detection system |
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