CN110846214A - Miniature multi-index nucleic acid analysis system and operation method thereof - Google Patents
Miniature multi-index nucleic acid analysis system and operation method thereof Download PDFInfo
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Abstract
The invention provides a miniature multi-index nucleic acid analysis system and an operation method thereof, which relate to the field of biological analysis and comprise a capillary array chip, a liquid-transferring gun, a plurality of reaction containers and a detection device; one end of the capillary array chip is used for extending into the reaction container, and the other end of the capillary array chip is communicated with the pipette head of the pipette; a plurality of primers for amplifying different nucleic acid indexes are arranged in the capillary array chip; the detection device is used for detecting the nucleic acid index in the capillary array chip, and the beneficial effects of the implementation of the invention are mainly as follows: can detect a plurality of indexes simultaneously, and can improve the number of the detected indexes by simply changing the arrangement mode of the capillary, thereby realizing the nucleic acid detection of multiple indexes; the capillary array chip and the pipette are small in size, the fluid is simple to control, high-flux nucleic acid detection can be realized, instant detection is realized, and the carrying is convenient.
Description
Technical Field
The invention relates to the field of biological analysis, in particular to a miniature multi-index nucleic acid analysis system and an operation method thereof.
Background
The nucleic acid detection steps can be mainly divided into nucleic acid extraction, amplification and detection, and the traditional nucleic acid detection has the defects of large manual operation, long time consumption and the like. By utilizing the micro-fluidic technology, various functional modules can be integrated on a tiny chip, and the gene analysis steps (DNA extraction, amplification and detection) are integrated and portable, so that the instant detection is realized. At present, the fluid control methods of microfluidic nucleic acid detection systems can be divided into two main categories: firstly, the method comprises the following steps: traditional microfluidic systems driven by mechanical pumps (air pumps, peristaltic pumps, syringe pumps, etc.). Secondly, the method comprises the following steps: a disk chip system driven by centrifugal force is disclosed.
Although the existing microfluidic nucleic acid detection system has good integration level and flux, the detection system still has large volume and high detection cost due to a plurality of design modules, so that the instant detection is difficult to realize.
Disclosure of Invention
The invention aims to solve the technical problems that the existing microfluidic nucleic acid detection system cannot meet the requirements of portability and multiple indexes at the same time, and has a complex structure and high processing cost.
In order to solve the above technical problems, the present invention discloses a micro multi-index nucleic acid analysis system, and the technical solution of the present invention is implemented as follows:
a micro multi-index nucleic acid analysis system driven by a pipette comprises a capillary array chip, a pipette, a plurality of reaction containers and a detection device; one end of the capillary array chip is used for extending into each reaction container, and the other end of the capillary array chip is communicated with the pipette head of the pipette; a plurality of primers for amplifying different nucleic acid indexes are arranged in the capillary array chip; the detection device is used for detecting the nucleic acid index in the capillary array chip.
Preferably, the capillary array chip comprises a plurality of capillaries, filter paper for nucleic acid extraction and bases, the filter paper is arranged at one end, close to the pipette tip, of each capillary, the bases coat the capillaries and the filter paper, one end, far away from the filter paper, of each base is communicated with the pipette tip, and the primers are arranged in the capillaries.
Preferably, the surface of the filter paper is modified by chitosan.
Preferably, the inner surface and the outer surface of the base are subjected to hydrophobic treatment.
Preferably, a sealing glue is arranged at the joint of the base and the capillary tube.
Preferably, the means of immobilization of the primer within the capillary comprises chitosan ligation, heating or freeze-drying techniques.
Preferably, one end of the capillary array chip is clamped in the pipette head.
Preferably, a sealing film is arranged at the joint of the capillary array chip and the pipette head of the pipette.
A method for operating a miniature multi-marker nucleic acid analysis system, comprising any one of the above miniature multi-marker nucleic acid analysis systems, the method comprising the steps of:
s1: putting three reagents, namely lysis solution, cleaning solution and amplification reaction agent into the three reaction containers respectively;
s2: putting the sample into a reaction container filled with lysate, and oscillating and mixing;
s3: immobilizing different ones of said primers within respective ones of said capillaries, each of said capillaries having immobilized therein a set of said primers;
s4: fixing the capillary array chip and the pipette head of the pipette;
s5: extending one end of the capillary array chip, which is far away from the pipette tip, into a reaction container filled with a lysate, sucking a reagent by the pipette, and discharging the reagent after a min;
s6: extending one end of the capillary array chip, which is far away from the pipette head, into a reaction container filled with cleaning liquid, sucking a reagent by the pipette, and discharging the reagent after b min;
s7: extending one end of the capillary array chip, which is far away from the pipette head of the pipette into a reaction container filled with an amplification reactant, and sucking a reagent by the pipette;
s8: separating the capillary array chip from the liquid-transfering gun, sealing by sealing glue, and heating;
s9: and placing the capillary array chip into the detection device for detection to obtain a detection result.
The beneficial effects of the implementation of the invention are as follows:
1. the three steps in the nucleic acid detection process, namely nucleic acid extraction, nucleic acid amplification and fluorescence detection, are integrated together, so that good integration is realized;
2. the capillary array chip can detect a plurality of indexes simultaneously, and the number of the detected indexes can be increased by simply changing the arrangement mode of the capillaries, so that the multi-index nucleic acid detection is realized;
3. the capillary array chip and the pipette are small in size, the fluid is simple to control, high-flux nucleic acid detection can be realized, the laboratory environment is broken away, the instant inspection is realized, the carrying is convenient, all materials and processing processes are low in cost, and the industrial production is easy to realize;
4. the capillary array chip utilizes the capillary as a nucleic acid amplification container, so that enzyme adsorption can be reduced, and the amplification efficiency is increased;
5. by using the multi-channel pipette, the operation of a plurality of capillary array chips can be realized, thereby realizing the analysis of a plurality of samples.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only one embodiment of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic cross-sectional view of a capillary array chip in accordance with an embodiment;
FIG. 2 is a schematic cross-sectional view of an embodiment utilizing a pipette gun for fluid control;
FIG. 3 is a flow chart of an embodiment detection process;
FIG. 4 is a schematic diagram of the distribution of primers in an embodiment.
In the above drawings, the reference numerals denote:
1-capillary array chip; 11-a capillary tube; 12-filter paper; 13-a base; 31-sealing glue; 32-a closing film; 4-a pipette; 5-a reaction vessel.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In a specific embodiment, a micro multi-index nucleic acid analysis system, as shown in fig. 1 and 2, comprises a capillary array chip 1, a pipette 4, a plurality of reaction vessels 5, and a detection device (not shown in fig. 3); one end of the capillary array chip is used for extending into the reaction container 5, and the other end is communicated with the gun head of the liquid-transfering gun 4; a plurality of primers (not shown in the figure) for amplifying different nucleic acid indexes are arranged in the capillary array chip; the detection device is used for nucleic acid indexes in the capillary array chip.
In the using process, a sample and lysis solution are placed in the reaction containers 5 to be mixed, the number of the reaction containers 5 can be determined according to the actual situation of the testing process, different reagents such as lysis solution or cleaning solution and the like are placed in each reaction container 5, no specific limitation is imposed on the reagents, the multi-index nucleic acid detection can be realized through different primers in the capillary array chip 1, and the nucleic acid extraction, amplification and detection are integrated. Pipette 4 rifle head and capillary array chip 1 intercommunication, can absorb the reagent in the reaction vessel 5 through pipette 4 and pass through capillary array chip 1, pipette 4 can be common single channel pipette 4 also can be multichannel pipette 4, and multichannel pipette 4 can realize the operation to a plurality of capillary array chips 1 to the realization is to the analysis of a plurality of samples.
Utilize pipette 4 to absorb and discharge reagent and carry out fluid control, the operator only need control pipette 4, add reagents such as sample, washing liquid and amplification reagent in proper order to capillary array chip 1, can realize the nucleic acid detection of many indexs, and detection system simple structure among the prior art relatively conveniently carries.
In the present embodiment, the detecting device is a detecting device in the prior art (such as a nucleic acid detecting instrument, etc.) and a detecting device that may be realized in the future, and a detecting device capable of realizing detection of nucleic acid can be used in the field, and is not specifically described herein.
In a preferred embodiment, as shown in fig. 2, one end of the capillary array chip 1 is embedded in the head of the pipette gun 4, and the capillary array chip 1 and the pipette gun 4 are connected in an embedded manner, so that the capillary array chip 1 and the pipette gun 4 can be detached, and the amplification reaction can be conveniently performed under corresponding experimental conditions or placed in a detection device for detection when the capillary array chip 1 is filled with the amplification reaction reagent, and other existing detachable connection methods are also applicable.
In a preferred embodiment, in order to further improve the connection tightness between the capillary array chip 1 and the pipette 4, a sealing film 32 is disposed at the joint between the capillary array chip 1 and the pipette tip 4, so as to reduce the reagent that enters the pipette 4 through the capillary array chip 1 from overflowing from the joint between the capillary array chip 1 and the pipette tip 4, and reduce the reagent loss, and besides the sealing film 32, other existing methods that can improve the connection tightness between the capillary array chip 1 and the pipette 4 can be used instead of the sealing film 32.
In a preferred embodiment, the capillary array chip 1 includes a plurality of capillaries 11, a filter paper 12 for nucleic acid extraction, and a base 13, the filter paper 12 is disposed at one end of the capillary 11 close to the pipette tip 4, the base 13 covers the capillary 11 and the filter paper 12, one end of the base 13 away from the filter paper 12 is communicated with the pipette tip 4, a reagent in the reaction container 5 sequentially passes through the capillary 11, the filter screen 12, and the base 13, and then reaches the pipette tip 4, and a primer is disposed in each capillary 11.
In this embodiment, 8 capillaries 11 are provided, which can realize nucleic acid detection of 8 different indexes, of course, according to different numbers of samples to be detected, corresponding numbers of capillaries 11 are used, 8 filter paper 12 is also provided corresponding to the capillaries 11, the filter paper 12 can be provided outside the end portion of the capillary 11, or can be provided in the capillary 11, the filter paper 12 is used for capturing the samples, the filter paper 12 can be made of glass fiber, etc., the base 13 is used for integrating the filter paper 12 and the capillary 11, the base 13 covers the filter paper 12 and the capillary 11, and can be partially covered or completely covered, which does not affect the reagent entering the capillary 11, and the setting of the base 13 can reduce the reagent overflow caused by the relative displacement of the filter paper 12 and the capillary 11 in the reagent flowing process.
The base 13 may be fabricated by any means such as micro-machining, machining or injection molding, and the base 13 may be fabricated by any one or two of the following mixed materials, such as silicon, ceramic, glass, and plastic, wherein the plastic includes: polyamide (PA), polybutylene terephthalate (PBT), Polycarbonate (PC), Polyethylene (PE), polymethyl methacrylate (PMMA), Polyoxymethylene (POM), polypropylene (PP), polystyrene diethyl ether (PPE), Polystyrene (PS), Polysulfone (PSU), polyether ether ketone (PEEK), Polydimethylsiloxane (PDMS), and the like.
The capillary tube 11 and the pipette 4 have small volume, the fluid control is simple, the capillary tube can be used for instant detection, the timeliness of nucleic acid detection can be improved, the detection of multiple indexes of nucleic acid can be realized by arranging different primers in the capillary tube 11, meanwhile, the arrangement mode of the capillary tube 11 is simply changed, the number of detection indexes can be improved, the capillary tube can be applied to the fields of infectious diseases, food safety and the like, the capillary tube has good universality, the fluid control is completely finished by the pipette 4, the operation is simple, the reliability is high, the carrying is convenient, when an amplification reactant enters the capillary tube 11, the capillary tube 11 is used as a container for nucleic acid amplification reaction, the enzyme adsorption can be reduced, the amplification efficiency is increased, all materials and processing technology are low in cost, and the industrial production.
In a preferred embodiment, the surface of the filter paper 12 is modified with chitosan (not shown), which can improve the adsorption force of the filter paper 12 on the sample particles when the sample passes through the surface of the filter paper 12, increase the sample adsorbed on the surface of the filter paper 12, further increase the amount of the sample entering the amplification reaction, reduce the loss of the sample during the operation, and improve the efficiency of nucleic acid extraction.
In a preferred embodiment, the inner and outer surfaces of the base 13 are both subjected to hydrophobic treatment, and the base 13 covers the filter paper 12 and the capillary tube 11, so that the hydrophobic treatment can reduce the reagent remaining in gaps of the filter paper 12 or the capillary tube 11, and the reagent may enter the tip of the pipetting gun 4 through the base 13 during the movement, the hydrophobic treatment can reduce the reagent adhering to the inner wall of the base 13 and reduce the reagent loss, the hydrophobic treatment can be realized by using a hydrophobic coating (Ultra-even Dry Top Coat) or the like, and other conventional hydrophobic treatment methods are all suitable without affecting the detection process.
In a preferred embodiment, a sealing adhesive 31 is disposed at the joint of the base 13 and the capillary tube 11, and the joint of the base 13 and the capillary tube 11 is sealed and fixed by the sealing adhesive 31, so as to improve the tightness of the joint between the capillary tube 11 and the base 13, reduce the gap between the capillary tube 11 and the base 13, reduce the circulation of air and reagents from the gap between the base 13 and the outer wall of the capillary tube 11, and improve the accuracy of the detection result.
In a preferred embodiment, the fixing manner of the primers in the capillary 11 includes chitosan connection, heating or freeze-drying technology, and the primers can be stably fixed on the inner wall of the capillary 11 by any one of the chitosan connection, heating or freeze-drying technology, so that the primers are prevented from entering the pipette 4 through the filter paper 12 along with the reagent entering the capillary 11, thereby affecting the extraction of the nucleic acid, and improving the accuracy of detecting each index of the nucleic acid to a certain extent.
The present invention also provides a method for operating a micro multi-index nucleic acid analysis system, as shown in fig. 3, including any one of the above micro multi-index nucleic acid analysis systems, the method comprising the steps of:
s1: putting three reagents of lysis solution, cleaning solution and amplification reaction agent into three reaction containers 5 respectively;
s2: putting the sample into a reaction container 5 filled with lysate, and oscillating and mixing;
s3: immobilizing different primers in each capillary 11, a set of the primers being immobilized in each capillary 11;
s4: fixing the capillary array chip 1 and a pipette head of a pipette 4;
s5: extending one end of the capillary array chip 1, which is far away from the gun head of the pipette 4, into a reaction vessel 5 filled with a lysate, sucking a reagent by the pipette 4, and discharging the reagent after a min;
s6: extending one end of the capillary array chip 1, which is far away from the gun head of the pipette 4, into a reaction vessel 5 filled with cleaning liquid, sucking a reagent through the pipette 4, and discharging the reagent after b min;
s7: extending one end of the capillary array chip 1, which is far away from the pipette head of the pipette 4, into a reaction vessel 5 filled with an amplification reactant, and sucking a reagent through the pipette 4;
s8: separating the capillary array chip 1 from the liquid-transfering gun 4, sealing by sealing glue, and heating;
s9: and (3) placing the capillary array chip 1 into the detection device for detection to obtain a detection result.
The detection process of different indexes of nucleic acid can be realized through the operation process of S1-S5, the extraction process of nucleic acid is realized through S6, the amplification reaction of nucleic acid is realized through S7 and S8, and the detection result is finally finished through S9, so that the micro-fluidic chip for detecting nucleic acid is realized, the extraction, amplification and detection of nucleic acid are integrated, the capillary array chip 1 in S8 uses the capillary as a container for the amplification reaction of nucleic acid, the enzyme adsorption can be reduced, and the amplification efficiency can be increased.
a min and b min need to be adjusted according to the amount of a sample and a reagent in the actual operation process, and b is preferably twice of a according to the operation condition, the action effect of the cleaning solution is better in the state, the capillary array chip 1 is provided with the outer diameter of 6mm and the height of 7mm, the volume of each capillary 11 is 2-2.5 microliter, the capillary array chip is used for containing an amplification reactant and can be placed in a 96-well plate to be matched with a detection device, and high-flux and portable nucleic acid detection is realized.
The reagents in the above embodiments are all generic and do not represent any particular component.
To make the present invention more comprehensible, an embodiment of a micro multi-index nucleic acid analysis system driven by a pipette is provided and described in detail as follows:
in this embodiment, the structure of the present invention is described in detail below, in which a chemically modified glass fiber filter paper 12 is used to adsorb DNA to extract nucleic acids, a loop-mediated isothermal amplification (LAMP) method is used to amplify nucleic acids, primers are fixed to the inner wall of a capillary 11 in advance by chemical modification, and calcein is used as a fluorescence indicator to perform fluorescence detection on the amplified products.
As shown in fig. 1 and 2, a capillary array chip (PAAC)1 includes 8 capillaries 11, 8 filter papers 12 and 1 base 13, wherein the filter papers are stored between the capillaries 11 and the bases 13 for capturing DNA, and in the present embodiment, the bases 13 are all manufactured by polymethyl methacrylate (PMMA) through machining or injection molding; the assembly method of the base 13 and the capillaries 11 is to use the sealant 31, each capillary 11 can fix a group of primers, in the implementation, according to the different number of the target of the sample to be detected, the corresponding number of capillaries 11 are used to realize the multi-index detection, as shown in fig. 4, the negative control in the figure is the capillary without any primer, and the nucleic acid detection is performed.
As shown in fig. 3, the reagent is injected and discharged by the air pressure control of the pipette 4; in practical implementation, firstly, a sample to be detected and lysis solution are mixed and then pass through a capillary array chip 1, and sample DNA is captured on filter paper 12; then, the cleaning solution flows through the capillary array chip 1 to wash and remove impurities such as protein except DNA; and finally, absorbing an amplification reagent into the capillary 11, separating the capillary array core 1 from the pipette head of the pipette 4, sealing the capillary array core in a 96-hole plate, heating the capillary array core 1 by using an external instrument, completing the amplification reaction, and detecting the amplification reaction by using a detection device.
The various embodiments listed above can be combined with each other without contradiction, and a person skilled in the art can combine the drawings and the above explanations of the embodiments as a basis for combining technical features of different embodiments.
It should be understood that the above-described embodiments are merely exemplary of the present invention, and are not intended to limit the present invention, and that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (9)
1. A beneficial miniature multi-index nucleic acid analysis system is characterized in that:
comprises a capillary array chip, a liquid-transfering gun, a plurality of reaction containers and a detection device;
one end of the capillary array chip is used for extending into the reaction container, and the other end of the capillary array chip is connected with the moving device
The liquid gun heads are communicated;
a plurality of primers for amplifying different nucleic acid indexes are arranged in the capillary array chip;
the detection device is used for detecting the nucleic acid index in the capillary array chip.
2. The miniature multi-marker nucleic acid analysis system of claim 1, wherein:
the capillary array chip comprises a plurality of capillaries, filter paper for nucleic acid extraction and bases, wherein the filter paper is arranged at one end, close to the pipette tip, of each capillary, the capillary and the filter paper are coated by the bases, one end, far away from the filter paper, of each base is communicated with the pipette tip, and the primers are arranged in the capillaries.
3. The miniature multi-marker nucleic acid analysis system of claim 2, wherein:
and performing chitosan modification on the surface of the filter paper.
4. The miniature multi-marker nucleic acid analysis system of claim 2, wherein:
and the inner surface and the outer surface of the base are subjected to hydrophobic treatment.
5. The miniature multi-marker nucleic acid analysis system of claim 2, wherein:
and a sealant is arranged at the joint of the base and the capillary tube.
6. The miniature multi-marker nucleic acid analysis system of claim 2, wherein:
the fixing mode of the primer in the capillary comprises chitosan connection, heating or freeze drying technology.
7. The miniature multi-marker nucleic acid analysis system of claim 1, wherein:
one end of the capillary array chip is clamped in the pipette head.
8. The miniature multi-marker nucleic acid analysis system of claim 7, wherein:
and a sealing film is arranged at the joint of the capillary array chip and the pipette head of the pipette.
9. A method of operating a miniature multi-index nucleic acid analysis system, comprising:
a miniature multi-marker nucleic acid analysis system comprising any of the preceding claims 2-8, and operating steps comprising:
s1: putting three reagents, namely lysis solution, cleaning solution and amplification reaction agent into the three reaction containers respectively;
s2: putting the sample into a reaction container filled with lysate, and oscillating and mixing;
s3: immobilizing different ones of said primers within respective ones of said capillaries, each of said capillaries having immobilized therein a set of said primers;
s4: fixing the capillary array chip and the pipette head of the pipette;
s5: extending one end of the capillary array chip, which is far away from the pipette tip, into a reaction container filled with a lysate, sucking a reagent by the pipette, and discharging the reagent after a min;
s6: extending one end of the capillary array chip, which is far away from the pipette head, into a reaction container filled with cleaning liquid, sucking a reagent by the pipette, and discharging the reagent after b min;
s7: extending one end of the capillary array chip, which is far away from the pipette head of the pipette into a reaction container filled with an amplification reactant, and sucking a reagent by the pipette;
s8: separating the capillary array chip from the liquid-transfering gun, sealing by sealing glue, and heating;
s9: and placing the capillary array chip into the detection device for detection to obtain a detection result.
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| CN112940922A (en) * | 2021-02-19 | 2021-06-11 | 清华大学 | Full-integrated pathogen nucleic acid detection micro-fluidic chip |
| CN114752658A (en) * | 2022-06-15 | 2022-07-15 | 上海邦先医疗科技有限公司 | Nucleic acid detection method and system based on micro-fluidic chip |
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