CN204162712U - The reaction unit of Visual retrieval nucleic acid constant-temperature amplification - Google Patents
The reaction unit of Visual retrieval nucleic acid constant-temperature amplification Download PDFInfo
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- CN204162712U CN204162712U CN201420491668.6U CN201420491668U CN204162712U CN 204162712 U CN204162712 U CN 204162712U CN 201420491668 U CN201420491668 U CN 201420491668U CN 204162712 U CN204162712 U CN 204162712U
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Abstract
The utility model discloses a kind of reaction unit of Visual retrieval nucleic acid constant-temperature amplification, comprise: but the liquid storage region of multiple separate connection, one of them region is used for carrying out nucleic acid constant-temperature amplification reaction, hold nucleic acid constant-temperature amplification reaction reagent, the all or part of reagent for holding for nucleic acid constant-temperature amplification reaction product Visual retrieval in other region, described reaction unit has the lid of described reaction vessel.In nucleic acid constant-temperature amplification process, nucleic acid amplification by product pyrophosphate is broken down into phosphate radical, after amplified reaction terminates, when remaining unchanged closed reactor, by all liquid mixing in reactor, whether there is colour-change in observing response device, whether meet the color that should embody after colouring reagents and phosphate radical react according to the liquid color in reactor and Visual retrieval is carried out to nucleic acid constant-temperature amplification.
Description
Technical field
The utility model belongs to chemical analysis, nucleic acid analysis, particularly relates to a kind of reaction unit of Visual retrieval nucleic acid isothermal amplification product.
Background technology
Along with the development of biotechnology, nucleic acid constant-temperature amplification technology is widely used in agricultural, medical science and field of food industry as novel nucleic acid amplification technologies.Isothermal amplification technology conventional at present has cross primer isothermal amplification technology (CPA), loop-mediated isothermal amplification technology (LAMP), strand displacement amplification (SDA), relies on the amplification (HDA) of helicase.Because such novel amplification technique can realize nucleic acid amplification (most of isothermal amplification technology desired reaction temperature is at 60 DEG C or about 37 DEG C) under lower isothermal condition, therefore the temperature control device of the precision required for normal PCR has been broken away from, greatly reduce cost, be subject to extensive concern.
For the method that nucleic acid constant-temperature amplification product detects, mainly contain electrophoresis, fluorescence dye, turbidity, immunity test strip etc. at present.Wherein, gel electrophoresis needs the operation of uncapping, and therefore easily causes Aerosol Pollution, and needs the fluorescence dye using high carcinogenic to be detrimental to health; Fluorometric assay needs special, huge, expensive opticinstrument; And although nephelometry can use naked-eye observation, require higher to nucleic acid amplification efficiency, require nucleic acid amplification output to reach minimum naked eyes visual turbidity and be easily subject to the impact of subjective factor, and be not suitable for all constant-temperature amplification detections.Therefore, this field needs to develop new detection method for nucleic acid amplification product.Visual retrieval has simply, directly, be convenient to the feature of large flux examination, can break away from complicated instrument facility, judge detected result.There is fluorexon fluorescence visual method at present, HNB method.These two kinds of methods utilize the concentration of metal ions of metallochromic indicator indirectly or directly in detection reaction system, thus colour-change occurs, and specifically, is become green from orange or become sky blue from purple respectively.Therefore, the method for current Visual retrieval nucleic acid amplification has inevitable shortcoming: 1. metallochromic indicator is easily subject to the impact of other metal ions in sample, causes error; 2. positive reaction result is from a kind of color to another color, is unfavorable for visual inspection; 3. detected object is the metal ion that in reaction solution, starting point concentration is higher, therefore can have higher background signal and reduce detection sensitivity.
Summary of the invention
The utility model first object is to provide a kind of reaction unit of Visual retrieval nucleic acid constant-temperature amplification, can realize when not opening lid, nucleic acid constant-temperature amplification is reacted can carry out uninterruptedly, the detection reaction of carrying out afterwards can normally be carried out according to the order of sequence, thus for Visual retrieval nucleic acid constant-temperature amplification provide equipment basis.For this reason, the utility model is by the following technical solutions:
A kind of reaction unit detected for visual nucleic acid constant-temperature amplification, comprise reaction vessel, but it is characterized in that described reaction vessel possesses the liquid storage region of multiple separate connection, one of them region is used for carrying out nucleic acid constant-temperature amplification reaction, hold nucleic acid constant-temperature amplification reaction reagent, the all or part of reagent (hereinafter referred to as product detection reagent) for holding for nucleic acid constant-temperature amplification reaction product Visual retrieval in other region, described reaction unit has the lid of described reaction vessel.The utility model reaction unit independent, to separate but the liquid storage region be communicated with ensures nucleic acid constant-temperature amplification reaction solution and product detection reagent separate, the different zones that is contained in device without interfering with each other before the reaction; When covered, by under operations such as acutely rocking or shake, put upside down, the liquid of different zones pre-stored just mixes for Visual retrieval.
Further, described reaction vessel is the test tube group that multiple test tube is connected as a single entity side by side, and in test tube group, the top of test tube is communicated with; The region of test tube below connection position is described liquid storage region.
The volume in the described liquid storage region for carrying out nucleic acid constant-temperature amplification reaction is less than or equal to 80ul and is more than or equal to 10ul.
Different reagent in the described reagent detected for visual nucleic acid constant-temperature amplification are placed in different liquid storage regions before detection reaction, the described reagent cumulative volume detected for visual nucleic acid constant-temperature amplification is preferably between 90 ~ 450ul, more preferably exist, between 90 ~ 270ul.
Described multiple quantity distinguishing the liquid storage region be still communicated with, being preferably 2-5, is more preferably 2-4.
Utilize reaction unit of the present utility model, can realize detecting nucleic acid constant-temperature amplification by indirect detection phosphate radical and detect, concrete grammar is as follows
Nucleic acid constant-temperature amplification reaction is carried out for the liquid storage space of nucleic acid constant-temperature amplification reaction in the reactor closed utilizing described reaction unit to provide, and the reagent carrying out nucleic acid constant-temperature amplification reaction product Visual retrieval is stored in other liquid storage region in described reactor one by one in advance, carry out containing reagent pyrophosphate being decomposed into phosphate radical in the reaction solution of nucleic acid constant-temperature amplification reaction, described in carry out nucleic acid constant-temperature amplification reaction product Visual retrieval reagent refer to and can carry out with phosphate radical the colouring reagents that reacts;
In nucleic acid constant-temperature amplification process, nucleic acid amplification by product pyrophosphate is broken down into phosphate radical, after nucleic acid constant-temperature amplification reaction terminates, when remaining unchanged closed reactor, by all liquid mixing in reactor, whether there is colour-change in observing response device and whether meet the color that should embody after colouring reagents and phosphate radical react according to the liquid color in reactor Visual retrieval is carried out to nucleic acid constant-temperature amplification.
Described nucleic acid constant-temperature amplification reaction reagent comprises nucleic acid constant-temperature amplification reaction mixture and inorganic pyrophosphatase; Wherein nucleic acid constant-temperature amplification reaction mixture can come from commercialization nucleic acid constant-temperature amplification kit, such as Guangzhou enlightening Australia PA101 Nos gene nucleic acid detection kit; Inorganic pyrophosphatase is the enzyme that tetra-sodium hydrolysis can be generated phosphate radical, is preferably the inorganic pyrophosphatase of heat resistant type.
Described nucleic acid constant-temperature amplification reaction can for amplification temperature be lower than the amplified reaction of 90 ° of C, and can be cross primer constant-temperature amplification (CPA), also can be loop-mediated isothermal amplification (LAMP).Described isothermal amplification reactions can be carried out under water-bath, metal bath or other heating conditions.
Different reagent in the described reagent detected for visual nucleic acid constant-temperature amplification are placed in different liquid storage regions before the reaction, and cumulative volume, preferably between 90 ~ 450ul, more preferably exists, between 90 ~ 270ul.The described reagent detected for visual nucleic acid constant-temperature amplification can be reductant solution and molybdate-antimonypotassium tartrate solution combination (combination one), or molybdate acid solution and malachite green solution combination (combination two), total volume, within above-mentioned volume range, has needs can supplement with water or Tris-HCl damping fluid.
In the described agent combination detected for visual nucleic acid constant-temperature amplification, the configuration of molybdate solution can use ammonium molybdate or Sodium orthomolybdate, preferred ammonium molybdate.
In the described agent combination detected for visual nucleic acid constant-temperature amplification, acid is bright sulfur acid, nitric acid, is preferably the vitriol oil.
In the described agent combination detected for visual nucleic acid constant-temperature amplification, reductant solution is configured by xitix or tin protochloride, is preferably xitix.
The utility model compared with prior art, the utility model provides a kind of nucleic acid isothermal amplification reaction conditions that can meet can meet again the reaction unit producing phosphate radical reaction and phosphate radical detection reaction condition, under can be implemented in the condition not opening reaction vessel, by mixing different solutions, detect the generation of phosphate radical in nucleic acid amplification reaction process, indirect visualization detects nucleic acid constant-temperature amplification: for negative sample, phosphate radical is not produced in whole testing process, therefore reaction solution is colourless, and for positive sample, along with the carrying out of amplified reaction, pyrophosphate constantly produces as by product, and phosphate radical is broken down under the effect of inorganic pyrophosphatase, thus react with colouring reagents and produce colour-change, reaction solution is in blue.Therefore the utility model has following characteristics, and first, the detection of nucleic acid amplification does not exist operation of uncapping, and can not cause crossed contamination; Secondly, make the detection method based on this reaction unit nucleic acid constant-temperature amplification, the detection that can realize from colourless to coloured judges, highly sensitive, be easy to the naked eye differentiate, and this detection judges the carrying out of nucleic acid amplification reaction by the generation of phosphate radical in detection amplification process, has lower background interference, be conducive to the accurate judgement to result.
Accompanying drawing explanation
Fig. 1 a is the schematic diagram of the reaction unit embodiment 1 detected for visual nucleic acid constant-temperature amplification
Fig. 1 b is the stereographic map of the reaction unit embodiment 1 detected for visual nucleic acid constant-temperature amplification.
Fig. 2 a is the schematic diagram of the reaction unit embodiment 2 detected for visual nucleic acid constant-temperature amplification.
Fig. 2 b is the stereographic map of the reaction unit embodiment 2 detected for visual nucleic acid constant-temperature amplification.
Fig. 3 a is the schematic diagram of the reaction unit embodiment 3 detected for visual nucleic acid constant-temperature amplification.
Fig. 3 b is the stereographic map of the reaction unit embodiment 3 detected for visual nucleic acid constant-temperature amplification.
Fig. 4 a is CPA constant-temperature amplification system positive and the negative sample visual test result contrast figure of example 1.
Fig. 4 b is LAMP constant-temperature amplification system positive and the negative sample visual test result contrast figure of example 5.
Embodiment
As Fig. 1 a, 1b, 2a, 2b, shown in 3a and 3b, the reaction unit detected for visual nucleic acid constant-temperature amplification provided by the utility model comprises reaction vessel 1, but described reaction vessel 1 possesses the liquid storage region of multiple separate connection, there are in embodiment 1 shown by Fig. 1 a and 1b three liquid storage regions and be respectively a, b, c, there are in embodiment 2 shown by Fig. 2 a and 2b four liquid storage regions and be respectively a, b, c, d, there are in embodiment 3 shown by Fig. 3 a and 3b five liquid storage regions and be respectively a, b, c, d, e, (such as region is a) for carrying out nucleic acid constant-temperature amplification reaction in one of them region, hold nucleic acid constant-temperature amplification reaction reagent, the all or part of reagent for holding for nucleic acid constant-temperature amplification reaction product Visual retrieval in other region, described reaction unit has the lid 10 of described reaction vessel.
In force, described reaction vessel 1 is the test tube group that multiple test tube is connected as a single entity side by side, and in test tube group, the top of test tube is communicated with; Test tube is described liquid storage region in the region at connection position less than 20.
Below in conjunction with above-mentioned reaction unit embodiment, the utility model is described in detail, but is not limited thereto.
Example 1: the utility model is used for visual cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.
Before reaction, reagent prepares: in a region of embodiment 1 reactor, adds 9ul reaction amplification liquid and (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Hangzhou You Sida biotech company transgenosis NOS sequence constant-temperature amplification kit, article No. I016-01), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); Ascorbic acid solution and the 8ul water of 2ul 10% are added in b region; 4ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 76ul water are added in c region.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C heating in water bath 1h.
Visual retrieval constant-temperature amplification: after having reacted, inverted device one, makes the liquid in 3 regions fully mix, judged result after 10min: the positive is blue (Fig. 4 a, 4), and feminine gender is colourless (Fig. 4 a, 3).
Example 2: the utility model is used for visual cross primer constant-temperature amplification (CPA) and detects transgenosis terminator T-Nos gene.
Nucleic acid constant-temperature amplification reaction is carried out: said apparatus is placed in 63 ° of C metal bath heating 1h.Other are with embodiment 1.
Example 3: the utility model is used for visual cross primer constant-temperature amplification (CPA) and detects streptococcus aureus.
Reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Yousida Biological Technology Co., Ltd., Hangzhou's streptococcus aureus constant-temperature amplification nucleic acid reagent box, article No. I011-01).Other are with embodiment 1.
Example 4: the utility model is used for visual cross primer constant-temperature amplification (CPA) and detects Salmonellas.
Reaction amplification liquid (includes primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Yousida Biological Technology Co., Ltd., Hangzhou's salmonella constant temperature amplification of nucleic acid test kit, article No. I047-01).Other are with embodiment 1.
Example 5: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.
In a region of embodiment 1 reactor, add 9ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia NOS gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA101S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); Ascorbic acid solution and the 8ul water of 2ul 10% are added in b region; 4ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 76ul water are added in c region.Other are with embodiment 1.
Visual retrieval constant-temperature amplification: after having reacted, puts upside down embodiment 1 reactor, the liquid in 3 regions is fully mixed, judged result after 10min: the positive is blue (Fig. 4 b, 4), and feminine gender is colourless (Fig. 4 b, 3).
Example 6: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects Salmonellas.
In a region of embodiment 1 reactor, add 19ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia Salmonellas kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA105S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); Ascorbic acid solution and the 16ul water of 4ul 10% are added in b region; 8ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 152ul water are added in c region.Other are with embodiment 1.
Example 7: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects streptococcus aureus.
In a region of embodiment 2 reactor, add 29ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia streptococcus aureus kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA109S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); Ascorbic acid solution and the 24ul water of 6ul 10% are added in b region; 12ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 108ul water are added in c region; 120ul water is added in d region.Other are with embodiment 1.
Example 8: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects Pseudomonas aeruginosa.
In a region of embodiment 2 reactor, add 39ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia pseudomonas aeruginosa kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA112S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); Ascorbic acid solution and the 32ul water of 8ul 10% are added in b region; 16ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 144ul water are added in c region; 160ul water is added in d region.Other are with embodiment 1.
Example 9: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects Pseudomonas aeruginosa.
In a region of embodiment 3 reactor, add 49ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia pseudomonas aeruginosa kit for detecting nucleic acid (constant temperature fluorescent method), article No. FA112S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); Ascorbic acid solution and the 40ul water of 10ul 10% are added in b region; 16ul ammonium molybdate-soluble tartrate antimony solution (comprising 21mM ammonium molybdate, 2mM soluble tartrate antimony, 5M sulfuric acid) and 144ul water are added in c region; 130ul water is added in d region; 130ul water is added in e region.Other are with embodiment 1.
Example 10: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.
In a region of embodiment 1 reactor, add 9ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia NOS gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA101S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); 5.875ul 80mM ammonium molybdate aqueous solution, the 2.5ul vitriol oil, 2ul 5mM malachite green solution and 29.625ul water are added in b region; 50ul water is added in c region.Other are with embodiment 1.
Example 11: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.
In a region of embodiment 1 reactor, add 19ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia NOS gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA101S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); 11.75ul 80mM ammonium molybdate aqueous solution, the 5ul vitriol oil, 4ul 5mM malachite green solution and 19.25ul water are added in b region; 140ul water is added in c region.Other are with embodiment 1.
Example 12: the utility model is used for visual loop-mediated isothermal amplification (LAMP) and detects transgenosis terminator T-Nos gene.
In a region of embodiment 2 reactor, add 29ul reaction amplification liquid and (include primer, dNTP, Tris-HCl damping fluid, Mg
2+, NH
4 +, template, from Guangzhou enlightening Australia NOS gene nucleic acid detection kit (constant temperature fluorescent method), article No. PA101S), and the heat resistant inorganic Pyrophosphate phosphohydrolase of 1ul 0.1U/ul (from New England Biolabs, article No. M0296s); 35.25ul 80mM ammonium molybdate aqueous solution, the 7.5ul vitriol oil, 6ul 5mM malachite green solution and 1.25ul water are added in b region; 110ul water is added in c region; 110ul water is added in d region.Other are with embodiment 1.
Claims (4)
1. the reaction unit detected for visual nucleic acid constant-temperature amplification, comprise reaction vessel, but it is characterized in that described reaction vessel possesses the liquid storage region of multiple separate connection, one of them region is used for carrying out nucleic acid constant-temperature amplification reaction, hold nucleic acid constant-temperature amplification reaction reagent, the all or part of reagent for holding for nucleic acid constant-temperature amplification reaction product Visual retrieval in other region, described reaction unit has the lid of described reaction vessel, to keep stopping property.
2. reaction unit as claimed in claim 1, is characterized in that described reaction vessel is the test tube group that multiple test tube is connected as a single entity side by side, and in test tube group, the top of test tube is communicated with; The region of test tube below connection position is described liquid storage region.
3. reaction unit as claimed in claim 1, is characterized in that the described volume for the liquid storage region of carrying out nucleic acid constant-temperature amplification reaction is less than or equal to 80ul and is more than or equal to 10ul.
4. reaction unit as claimed in claim 1, is characterized in that the liquid storage amount of the overall adaptation 90 ~ 450ul in the liquid storage region of the described reagent detected for visual nucleic acid constant-temperature amplification.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967790A (en) * | 2017-02-20 | 2017-07-21 | 浙江大学 | A kind of detection method of nucleic acid amplification |
| CN111057749A (en) * | 2019-11-22 | 2020-04-24 | 福州大学 | Visual constant-temperature amplification product detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967790A (en) * | 2017-02-20 | 2017-07-21 | 浙江大学 | A kind of detection method of nucleic acid amplification |
| CN106967790B (en) * | 2017-02-20 | 2020-08-25 | 浙江大学 | Detection method for nucleic acid amplification |
| CN111057749A (en) * | 2019-11-22 | 2020-04-24 | 福州大学 | Visual constant-temperature amplification product detection method |
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