CN104894277B - Method for preventing aerosol pollution of isothermal amplification product of nucleic acid - Google Patents
Method for preventing aerosol pollution of isothermal amplification product of nucleic acid Download PDFInfo
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- CN104894277B CN104894277B CN201510342799.7A CN201510342799A CN104894277B CN 104894277 B CN104894277 B CN 104894277B CN 201510342799 A CN201510342799 A CN 201510342799A CN 104894277 B CN104894277 B CN 104894277B
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to a method for preventing aerosol pollution of isothermal amplification products of nucleic acid. Fixing the PCR single tube filled with the reaction liquid by using a PCR single tube clamp, or fixing the PCR eight-tube filled with the reaction liquid by using a PCR eight-tube clamp, penetrating the PCR single tube clamp or the PCR eight-tube clamp onto a tube frame of a water bath kettle, pressing down the tube frame, immersing the PCR single tube or the PCR eight-tube filled with the reaction liquid below the liquid level of the water bath kettle, carrying out nucleic acid isothermal amplification at a set temperature, and judging an amplification result through the color change of an indicator after the reaction is finished. By performing isothermal amplification of nucleic acid by this method, the amplification product is dissolved in water by the occasionally leaked gas through water washing, and aerosol contamination caused by the nucleic acid amplification product can be prevented.
Description
Technical Field
The invention relates to a method for preventing aerosol pollution of isothermal amplification products of nucleic acid, belonging to the technical field of biology.
Background
Isothermal amplification techniques of nucleic acids include loop-mediated isothermal amplification (LAMP), nucleic acid sequence dependent amplification (NASBA), strand displacement amplification reaction (SDA), rolling circle amplification Reaction (RCA), and melting enzyme amplification (HAD), etc., which are time-consuming without temperature change, do not require expensive instruments, and have a tendency to replace the chain polymerase reaction (PCR). However, aerosol pollution of the amplification product is still a limiting factor for the popularization and application of the isothermal nucleic acid amplification technology. Currently, aerosol pollution is reduced by commonly using uncapping detection of amplification products, for example, Hangzhou Yosida biotechnology limited develops a totally enclosed target nucleic acid amplification product rapid detection device (patent number: ZL 200610109620.4), Boao biology limited develops a constant temperature amplification microfluidic chip, Huafeng biotechnology limited adopts an HF reaction tube to realize uncapping detection, and the existing detection method either obviously increases the detection cost or cannot completely solve the aerosol pollution problem. Therefore, the method for cheaply and effectively preventing the aerosol pollution of the isothermal amplification product of the nucleic acid is very important for the popularization and application of the isothermal amplification technology of the nucleic acid.
Disclosure of Invention
The invention aims to provide a method for preventing aerosol pollution of isothermal amplification products of nucleic acid, which can rapidly and effectively promote the popularization and application of isothermal amplification technology of nucleic acid without increasing the cost of consumables.
A method for preventing aerosol pollution of isothermal amplification products of nucleic acid is characterized in that nucleic acid is amplified in an underwater water bath at constant temperature, and occasionally leaked gas dissolves the amplification products in water through water washing so as to prevent aerosol pollution caused by the nucleic acid amplification products.
The method is realized through the following accessories: the fittings comprise a PCR single-tube pipe clamp or a PCR eight-tube pipe clamp, and a pipe support for fixing the PCR single-tube pipe clamp or the PCR eight-tube pipe clamp.
The method comprises the following operation steps: fixing the PCR single tube filled with the reaction liquid by using a PCR single tube clamp, or fixing the PCR eight-tube filled with the reaction liquid by using a PCR eight-tube clamp, penetrating the PCR single tube clamp or the PCR eight-tube clamp onto a tube frame of a water bath kettle, pressing down the tube frame, immersing the PCR single tube or the PCR eight-tube filled with the reaction liquid below the liquid level of the water bath kettle, carrying out nucleic acid isothermal amplification at a set temperature, and judging an amplification result through the color change of an indicator after the reaction is finished.
The invention has the advantages that: under the condition of not increasing the cost of detection consumables, the aerosol pollution of nucleic acid isothermal amplification products can be effectively controlled, and the popularization and application of the nucleic acid isothermal amplification technology are rapidly promoted.
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FIG. 1: the PCR single-tube clamp structure of the invention is schematically shown.
Detailed Description
Example 1: salmonella bacteria (A), (B)Salmonella) Specificity groupDue to the fact thatinvAThe underwater LAMP amplification detection method comprises the following steps:
(1) reaction system (reaction total volume 25 mu l)
(2) Amplification reaction
Fixing the PCR single tube filled with the reaction liquid by using a PCR single tube clamp, or fixing the PCR eight-tube filled with the reaction liquid by using a PCR eight-tube clamp, penetrating the PCR single tube clamp or the PCR eight-tube clamp onto a tube frame of a water bath kettle, pressing down the tube frame, immersing the PCR single tube or the PCR eight-tube filled with the reaction liquid below the liquid level of the water bath kettle, and carrying out thermostatic water bath at 60 ℃ for 50 minutes, wherein the reaction liquid is negative if the reaction liquid is light yellow; if the reaction solution turns green, it is positive.
Example 2: trypanosoma brucei (I)Trypanosoma brucei gambiense) The underwater LAMP amplification detection method of the gene fragment 5.8S-ITS2 comprises the following steps:
(1) reaction system (reaction total volume 25 mu l)
(3) Amplification reaction
Fixing the PCR single tube filled with the reaction liquid by using a PCR single tube clamp, or fixing the PCR eight-tube filled with the reaction liquid by using a PCR eight-tube clamp, penetrating the PCR single tube clamp or the PCR eight-tube clamp onto a tube frame of a water bath kettle, pressing down the tube frame, immersing the PCR single tube or the PCR eight-tube filled with the reaction liquid below the liquid level of the water bath kettle, and carrying out thermostatic water bath at 60 ℃ for 50 minutes, wherein the reaction liquid is negative if the reaction liquid is light yellow; if the reaction solution turns green, it is positive.
Finally, it should be noted that: the above examples are intended to illustrate, not to limit, the present invention, and it will be understood by those skilled in the art that: any modification or partial replacement within the spirit and scope of the present invention should be covered by the appended claims.
Claims (1)
1. A method for preventing aerosol pollution of isothermal amplification products of nucleic acid is characterized in that a PCR single tube or a PCR eight-connecting tube filled with reaction liquid is placed in an underwater water bath for amplifying the nucleic acid at constant temperature, and occasionally leaked gas is washed to dissolve the amplification products in the water, so that the aerosol pollution caused by the nucleic acid amplification products is prevented; judging the amplification result through the color change of the indicator after the isothermal amplification reaction of the nucleic acid is finished; the temperature of the constant temperature amplified nucleic acid is 60 ℃; operating to place the PCR monotube or PCR octal tube under water according to the following steps: fixing the PCR single tube filled with the reaction liquid by using a PCR single tube clamp, or fixing the PCR eight-tube filled with the reaction liquid by using a PCR eight-tube clamp, penetrating the PCR single tube clamp or the PCR eight-tube clamp onto a tube frame of the water bath kettle, pressing down the tube frame, and immersing the PCR single tube or the PCR eight-tube filled with the reaction liquid below the liquid level of the water bath kettle.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510342799.7A CN104894277B (en) | 2015-06-19 | 2015-06-19 | Method for preventing aerosol pollution of isothermal amplification product of nucleic acid |
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| CN201510342799.7A CN104894277B (en) | 2015-06-19 | 2015-06-19 | Method for preventing aerosol pollution of isothermal amplification product of nucleic acid |
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| CN104894277A CN104894277A (en) | 2015-09-09 |
| CN104894277B true CN104894277B (en) | 2020-09-11 |
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| CN108588196B (en) * | 2018-04-23 | 2021-12-14 | 北京中能通达科技发展中心（有限合伙） | Method for preventing aerosol pollution formed by PCR |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1415381A (en) * | 2002-09-26 | 2003-05-07 | 陈�光 | Method for eliminating pollution of nucleic acid |
| CN101851652A (en) * | 2010-04-27 | 2010-10-06 | 上海交通大学 | General multiplex polymerase chain reaction realization method based on microarray chip |
| CN103421676A (en) * | 2012-05-25 | 2013-12-04 | 国家纳米科学中心 | Nucleic acid isothermal amplification reaction system, and making method and applications thereof |
| CN103630518A (en) * | 2013-11-18 | 2014-03-12 | 蔡典其 | Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103725600A (en) * | 2012-10-16 | 2014-04-16 | 哈尔滨德歌生物科技有限公司 | Negative-pressure reaction tube |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1415381A (en) * | 2002-09-26 | 2003-05-07 | 陈�光 | Method for eliminating pollution of nucleic acid |
| CN101851652A (en) * | 2010-04-27 | 2010-10-06 | 上海交通大学 | General multiplex polymerase chain reaction realization method based on microarray chip |
| CN103421676A (en) * | 2012-05-25 | 2013-12-04 | 国家纳米科学中心 | Nucleic acid isothermal amplification reaction system, and making method and applications thereof |
| CN103630518A (en) * | 2013-11-18 | 2014-03-12 | 蔡典其 | Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method |
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