CN111004719B - Nucleic acid detection module, detection unit and detection system - Google Patents
Nucleic acid detection module, detection unit and detection system Download PDFInfo
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Abstract
The invention discloses a nucleic acid detection module, a detection unit and a detection system. The nucleic acid detection module comprises a pretreatment component and a microfluidic detection component; the pretreatment assembly comprises a nucleic acid extraction element and a main body module, wherein the main body module comprises a washing cavity and a reaction cavity, the washing cavity is matched with the nucleic acid extraction element, the washing cavity is used for containing a washing reagent, the washing reagent is used for purifying the nucleic acid, and the reaction cavity is used for containing a nucleic acid amplification reaction system capable of amplifying the nucleic acid to form an amplicon; the microfluidic detection assembly comprises a microfluidic channel, the microfluidic channel is connected with the reaction cavity, the microfluidic channel is used for accommodating a detection reaction system containing amplicons and auxiliary detection reagents, and the detection reaction system can generate a detectable signal. According to the invention, the pretreatment component and the microfluidic component are combined, the iRPAS signal amplification method is adopted to amplify the extracted nucleic acid, and the intelligent mobile phone is used to heat the amplification process, so that the full integration of sample collection, nucleic acid release, purification, amplification and detection is realized.
Description
Technical Field
The invention particularly relates to a nucleic acid detection module, a detection unit and a detection system, and belongs to the technical field of microfluidic detection.
Background
Nucleic acid detection is of great significance in life sciences and is widely applied to clinical diagnosis, agricultural monitoring, food safety and plant pathogen infection detection. Furthermore, with the deep understanding of human healthy genome information, various types of nucleotide mutations such as gene replacement, insertion and deletion have been proved to be closely related to diseases such as genetic diseases, metabolic diseases and cancers, and even Single Nucleotide Polymorphisms (SNPs) have important relevance for individual disease susceptibility. Thus, there is an urgent need for a nucleic acid detection method having high sensitivity and Shan Jianji specificity. Traditional nucleic acid detection methods such as DNA microarrays, DNA sequencing, multiplex ligation-dependent probe amplification, reverse dot blotting, high resolution melting, and some sensor-based methods, rely on expensive equipment and modern laboratory environments, and prevent their use in on-site, point-of-care nucleic acid detection in non-laboratory environments.
The instant detection is a detection means and trend which rapidly develop in recent years, namely a technical method for rapidly completing sample collection, pretreatment, detection reaction and result reading and distinguishing in the field. The method has the characteristics of rapidness, simplicity, convenience, high integration, no dependence on large-scale instruments and equipment, professional staff and the like. The instant detection provides possibility for public health monitoring of basic units with limited resources, household health care, disease prevention, and on-site rapid detection of food, water resources, environment and the like in underdeveloped areas. In recent years, many innovative detection methods have been developed in nucleic acid detection, particularly isothermal nucleic acid amplification, for example, loop-mediated isothermal amplification methods, rolling circle amplification methods, recombinase polymerase amplification methods, and the like, and are expected to be widely used in the detection of nucleic acids in the future.
The 3D printing microfluidic technology utilizes the 3D printing technology to directly print a microfluidic chip or prepare an emerging technology of a microfluidic chip die, so that the application range of the microfluidic is further promoted and extended. Microfluidic (Microfluidics) is a technology which appears in nineties of the last century, and is used for operating and controlling fluid on a micrometer scale, and has the characteristics of small volume, large specific surface area, small reagent dosage, high flux, high reaction speed and the like, and has wide prospect in the field of instant detection. However, the preparation of the microfluidic chip depends on a semiconductor process, the processing process is numerous, the preparation needs to be completed in an ultra-clean room, and the application range of the microfluidic chip is limited. In recent years, along with the development of 3D printing technology, it is also possible to manufacture a microfluidic chip by using the 3D printing technology, and the 3D printing microfluidic chip has the advantages of simple manufacture, low processing cost and the like, and is rapidly developed in the field of biomedical detection.
For example, a microfluidic chip for visualizing the immediate detection of pathogen nucleic acids is described in CN107904161 a. The chip is provided with a sample processing area, a DNA extraction area, a fluid control area and a visual detection area which are sequentially communicated, nucleic acid extraction is carried out by using a magnetic bead method, and nucleic acid amplification is carried out by using a loop-mediated isothermal amplification method. The microfluidic chip integrates sample processing, pathogen nucleic acid extraction, amplification and visual detection. However, the chip operation still needs to dilute the sample with normal saline, the centrifugal operation needs to be performed by using a magnetic bead and a pipettor for auxiliary operation, and more importantly, the reaction still needs to be heated by a heat table with constant temperature of 60-68 ℃, so that the on-site instant detection cannot be completely realized. And, it adopts traditional photoetching micro-fluidic chip manufacturing process, and whole runner is the micron level, can't carry out liquid injection through the manual work for its easy and simple to handle nature drops greatly, and the pretreatment of sample needs the normal saline dilution and centrifuge centrifugation just can concentrate the sample, adopts the magnetic bead method to need the magnetic column to assist, and reagent application of sample needs to use the pipettor, and whole operation process is limited by micro-fluidic chip's disadvantage: the manual operation is not easy.
The invention provides a palm-type universal microfluidic chip instant detection device and application thereof in CN103308502B, which mainly adopts a fluorescence loop-mediated isothermal amplification method to amplify and detect nucleic acid, integrates a mobile power supply heating system and a fluorescence detection system, overcomes the defects that the traditional nucleic acid detection reaction needs external power supply and external large-scale computer, greatly expands the applicable range of the device, and is matched with an application program (APP) running on intelligent mobile equipment, easy to operate and applicable to nucleic acid amplification reaction; however, the scheme does not have a sample pretreatment system, the detected sample is an enriched and purified sample, and the on-site instant detection cannot be completely realized by the aid of a nucleic acid enrichment and purification system; the nucleic acid immediate detection device adopts a palm microfluidic chip, adopts a fluorescence loop-mediated isothermal amplification method to amplify and detect nucleic acid, and integrates a mobile power supply heating system and a fluorescence detection system; however, the method does not have a sample pretreatment system, the detected sample is a concentrated and purified sample, the on-site instant detection cannot be truly realized, and the specificity of the method cannot reach the single base difference level.
In US9469871B 2a method and apparatus for on-the-fly nucleic acid amplification and detection is disclosed, embodiments of which include a fully integrated sample-to-result molecular diagnostic instrument, preferably using a loop-mediated isothermal amplification method, to reduce the instrument requirements associated with nucleic acid amplification and to enable detection of target amplification by colour shift or fluorescence by adding a dye to the amplification reaction. However, this method still requires an external heating device to supply the heat required for the reaction (60-65 ℃), and the detection specificity does not achieve a single base difference. The whole detection device is in a macroscopic millimeter scale, and a microfluidic chip cannot be realized; the method is mainly based on a syringe and matched devices to purify and detect nucleic acid on a macroscopic scale, and the method also adopts a loop-mediated isothermal amplification method, the reaction needs to be carried out at 60-65 ℃, an additional heating device is needed for assistance, and the specificity of the method cannot reach the single base difference level.
Disclosure of Invention
The invention mainly aims to provide a nucleic acid detection module, a detection unit and a detection system, which are used for overcoming the defects in the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a nucleic acid detection module, which comprises a preprocessing component (110) and a microfluidic detection component (120);
wherein the preprocessing component (110) comprises:
a nucleic acid extraction element for extracting at least nucleic acids in a sample;
a body module (1103) comprising a wash chamber (11031) cooperating with the nucleic acid extraction element and a reaction chamber (11032), the wash chamber (11031) for housing a wash reagent for purifying the nucleic acid, the reaction chamber (11032) for housing a nucleic acid amplification reaction system capable of amplifying the nucleic acid to form an amplicon;
the microfluidic detection assembly (120) comprises a microfluidic channel connected to a reaction chamber (11032) for housing a detection reaction system comprising the amplicon, an auxiliary detection reagent, the detection reaction system being capable of generating a detectable signal.
Further, the nucleic acid extraction element comprises a filter basket (1101), the filter basket (1101) is provided with a top port (11012) and a bottom port (11013), a DNA extraction test paper (11011) is arranged in the filter basket (1101), the top port (11012) is used for injecting the washing reagent into the filter basket (1101), and the bottom port (11013) is used for enabling a sample to enter the filter basket (1101) and be in contact with the DNA extraction test paper (11011).
Further, when the filter basket (1101) is partially inserted into the washing chamber (11031), the washing reagent supplied from the washing reagent supply pipe can enter the washing chamber (11031) through the inner chamber of the filter basket (1101).
Preferably, the washing reagent supply tube has an air gap therein to separate different washing reagents from each other.
Preferably, the bottom of the washing cavity (11031) is provided with a liquid discharge hole (110311).
Further, when the filter basket (1101) is partially inserted into the reaction chamber (11032), the nucleic acid adsorbed on the DNA extraction test paper (11011) can be matched with other components required by the nucleic acid amplification reaction in the reaction chamber (11032) to form a nucleic acid amplification reaction system, so that amplicons are formed.
Further, the filter basket (1101) is provided with a connecting part (11014) matched with the valve (1102), the reaction cavity (11032) is provided with a connecting pipe (110321) communicated with the microfluidic detection assembly, when the filter basket (1101) is connected with the reaction cavity (11032) through the valve (1102), and the valve (1102) is in an open state, the inner cavity of the filter basket (1101) is communicated with the pipeline (110321) through a liquid outlet hole on the valve (1102).
Further, the valve (1102) has an insertion hole (11024) and a discharge hole (11023), and when the filter basket (1101) is partially inserted into the insertion hole (11024), the inner cavity of the filter basket (1101) communicates with the discharge hole (11023).
Further, the valve (1102) also has a groove (11021) for mounting a sealing ring.
Further, the valve (1102) is also provided with a rotary positioning mark (11022), and the reaction cavity (11032) is also provided with a positioning groove (110322) matched with the rotary positioning mark (11022) of the valve (1102)
Further, at least one connecting protrusion (11033) and/or at least one connecting groove (11034) are arranged on one main body module, and the at least one connecting protrusion (11033) and the at least one connecting groove (11034) are matched with the corresponding at least one connecting protrusion (11033) and the corresponding at least one connecting groove (11034) on the other main body module respectively.
Further, the auxiliary detection reagent comprises a nano-gold buffer solution with streptavidin modification, and the nucleic acid amplification reaction system comprises a tailed forward primer and a biotinylated reverse primer.
Further, the auxiliary detection reagent also comprises a mixed solution of silver salt and an initiator.
In some more specific embodiments, the microfluidic detection assembly (120) further comprises a temporary probe channel in communication with the microfluidic channel, the temporary probe channel for providing at least a probe complementary to an amplicon tail base derived from the tailed forward primer.
Further, the microfluidic channel and the temporary probe channel are respectively distributed in a microfluidic channel layer (1201) and a temporary probe channel layer (1202) of the microfluidic detection assembly (120).
Further, the preprocessing component (110) includes a 3D printing component.
Further, the detection reaction system comprises an iRAS amplification detection reaction system.
Further, the detectable signal comprises a visual detection signal.
The embodiment of the invention also provides a nucleic acid detection unit, which comprises:
the intelligent terminal is at least used for processing the detectable signal output by the nucleic acid detection module.
Furthermore, the intelligent terminal is also used for providing heat required for facilitating the reaction for a nucleic acid amplification reaction system and a detection reaction system which are carried out in the nucleic acid detection unit.
Further, the intelligent terminal is internally provided with a heating module.
Further, the intelligent terminal comprises a mobile phone.
In some more specific embodiments, the nucleic acid detection unit further comprises a thermal container mated with the nucleic acid detection module, the thermal container having a temperature display strip and a temperature quality control disk.
The embodiment of the invention also provides a nucleic acid detection system, which comprises:
the nucleic acid detection module or the nucleic acid detection unit; and
the intelligent terminal adaptation module comprises a foldable support, wherein the foldable support is provided with an inserting port (2301) and a detection signal taking port (2302), when the nucleic acid detection module is inserted into the foldable support from the inserting port (2301), and a signal taking mechanism of the intelligent terminal is arranged at the detection signal taking port (2302), the intelligent terminal can collect a detectable signal in the nucleic acid detection module.
Further, the foldable stand is a foldable paper stand (230).
Further, the signal pickup mechanism comprises a camera, and an optical magnifier (210) matched with the camera is further arranged at the detection signal pickup port (2302).
Further, the signal pickup mechanism comprises a camera, and a light source is further arranged at a position corresponding to the detection signal pickup port (2302).
Compared with the prior art, the nucleic acid instant detection system provided by the invention combines the pretreatment component and the microfluidic component, adopts the iRPAS signal amplification method to amplify the extracted nucleic acid, and uses the smart phone to heat the amplification process, thereby realizing the full integration of sample collection, nucleic acid release, purification, amplification and detection.
Drawings
FIG. 1a is a schematic diagram showing the structure of a nucleic acid detecting module according to an exemplary embodiment of the present invention;
FIG. 1b is a top view of a nucleic acid detection module according to an exemplary embodiment of the present invention;
FIG. 1c is a schematic illustration of a filter basket according to an exemplary embodiment of the present invention;
FIG. 1d is a schematic illustration of a rotary valve according to an exemplary embodiment of the present invention;
FIG. 2a is a schematic illustration of a folded structure of a foldable paper holder according to an exemplary embodiment of the present invention
FIG. 2b is a schematic illustration of a foldable paper stand in an exemplary embodiment of the invention, shown disassembled;
FIG. 2c is a schematic view of a folding flow of a foldable stand according to an exemplary embodiment of the present invention, wherein broken lines are folding lines;
FIG. 3a shows the results of detecting the type SEA of alpha thalassemia (long fragment deletion) by using the instant nucleic acid detection system provided by the invention;
FIG. 3b shows the results of detecting type CD41 (short fragment deletion) of thalassemia beta using the instant nucleic acid detection system of the present invention;
FIG. 3c shows the detection result of the type CD17 (single base substitution) of beta thalassemia by using the instant nucleic acid detection system provided by the invention;
FIG. 3d shows the detection result of the type CD71 (single base insertion) of beta thalassemia by using the instant nucleic acid detection system provided by the invention;
FIG. 4a shows the detection result of E.coli (E.coli) in a urine sample using the instant nucleic acid detection system of the present invention;
FIG. 4b is a sample of Klebsiella pneumoniae (K.pn) in a urine sample using the instant nucleic acid detection system of the present invention;
FIG. 4c shows the detection result of E.coli (E.coli) in a milk sample using the instant nucleic acid detection system of the present invention;
FIG. 4d shows the detection result of E.coli (E.coli) in a river water sample by using the instant nucleic acid detection system provided by the invention;
FIG. 4e shows the detection result of the instant nucleic acid detection system provided by the invention for detecting the kiwi fruit canker pathogen (P.sa;
FIG. 5 shows the intensity of detection signals obtained by detecting nucleic acids using the nucleic acid detection system according to the embodiment of the present invention under different environmental temperature conditions;
FIG. 6 is a graph showing the correlation between the concentration of target DNA and the intensity of the detected signal in an exemplary embodiment of the present invention;
FIG. 7 is a graph showing the temperature effects of a smart phone heating nucleic acid detection module under different ambient air temperature conditions;
FIG. 8 is a schematic view of a thermal insulation bag according to an exemplary embodiment of the present invention;
FIG. 9 is a diagram of a mobile phone interface for a heating application or program in a smart phone in accordance with an exemplary embodiment of the present invention;
fig. 10 is a block diagram of a control flow for controlling the number of algorithms to be run to regulate the heat of a smart phone in an exemplary embodiment of the present invention.
Detailed Description
In view of the shortcomings in the prior art, the inventor of the present invention has long studied and practiced in a large number of ways to propose the technical scheme of the present invention. The technical scheme, the implementation process, the principle and the like are further explained as follows.
Referring to fig. 1 a-1 d, a nucleic acid real-time detection system according to an exemplary embodiment of the present invention is mainly composed of a detection chip (i.e., the aforementioned nucleic acid detection module, hereinafter the same) and a customized smart mobile device 200, which can be put in a pocket by an end user and quickly assembled for use in a limited resource situation.
Referring to fig. 1a to 1d, the detection chip mainly includes a preprocessing component 110 and a microfluidic detection component 120; the pre-processing assembly 110 is 3D printed and includes a filter basket 1101 for sample absorption and DNA purification, a rotary valve (i.e., the aforementioned valve, supra) 1102 for controlling the opening and closing of the microfluidic channels, and a body module 1103 for constructing the chambers and connecting the detection assembly.
The filter basket 1101 has a top port 11012 and a bottom port 11013, a DNA extraction test paper 11011 is provided inside, a seal ring placement groove 11021 is provided on the outer surface of the rotary valve 1102, and a rotary positioning mark 11022 and a side wall liquid outlet hole 11023 are provided on the bottom.
The main body module 1103 has a washing chamber 11031 and a reaction chamber 11032, the bottom of the washing chamber has a liquid discharge hole 110311, the side of the reaction chamber is connected with the microfluidic detection assembly through a connecting pipe (110321), the bottom is provided with a positioning groove 110322 for positioning a rotary valve, in addition, at least one connecting protrusion 11033 and/or at least one connecting groove 11034 are provided on one main body module, and the at least one connecting protrusion 11033 and the at least one connecting groove 11031 are respectively matched with the corresponding at least one connecting protrusion and the corresponding at least one connecting groove on the other main body module.
Next, the microfluidic detection assembly 120 includes a microfluidic channel layer 1201 made of Polydimethylsiloxane (PDMS) and an aldehyde-functionalized and modified glass substrate, the microfluidic channels are distributed in the microfluidic channel layer 1201, and DNA or RNA probes capable of complementarily binding to the local sequence of the amplicon are mounted on the surface of the probe region of the aldehyde-functionalized and modified glass substrate.
Specifically, the temporary probe channel layer 1202 and the glass substrate modified by aldehyde group functionalization are combined through an aldehyde-amine condensation reaction, a detection probe is injected, the temporary probe channel layer 1202 is peeled off after the surface of the glass substrate modified by aldehyde group functionalization is coated, and the microfluidic channel layer 1201 and the glass substrate modified by aldehyde group functionalization are combined through an aldehyde-amine condensation reaction to form the final microfluidic detection assembly 120.
In particular, the temporary probe channel is used for immobilizing different probes on different areas of the microfluidic channel; according to the embodiment of the invention, different nucleic acid probes can be immobilized in different areas of the microfluidic channel in advance; for example, probes may be immobilized in specific locations of the microfluidic channel by printing, etc., or temporary probe channels may be immobilized in different areas of the microfluidic channel to immobilize different nucleic acid probes.
Specifically, the rotary valve 1102 in this embodiment is a sleeve with a liquid outlet on its side wall, and the filter basket 1101 is inserted into the rotary valve (sleeve) 1102 and then together into the reaction chamber 11032; since the liquid outlet holes on the side wall of the rotary valve 1102 are staggered from the holes on the side wall of the reaction chamber 11032, the reaction liquid can remain in the reaction chamber 11032, but the rotary valve 1102 is not required, and the filter basket 1101 can be directly inserted into the reaction chamber 11032 with the reaction liquid, at this time, although the side wall of the reaction chamber 11032 is provided with a connecting hole and an external connecting pipe 110321 which are connected with the microfluidic pipeline, the liquid is not easy to flow into the microfluidic pipeline, because the pipe diameter of the microfluidic pipeline is small, and a certain pressure is required for the liquid to enter the microfluidic pipeline as a thrust force; the rotary valve 1102 is used only to better stop the reaction solution from leaking into the microfluidic channel in advance during the reaction.
Specifically, the connecting pipe 110321 is communicated with the reaction cavity through a connecting hole on the side wall of the reaction cavity, when the liquid outlet hole 11023 is misplaced with the connecting hole, the solution in the filter basket 1101 is not communicated with the connecting pipe 110321, the solution in the reaction cavity 11032 cannot flow outwards, and the reaction is carried out in the reaction cavity 11032; after the reaction is completed, the whole rotary valve 1102 is rotated to enable the liquid outlet hole 11023 to be opposite to the connecting hole, at the moment, the reaction cavity 11032 is communicated with the connecting pipe 110321, air is pushed in from the valve 11012, and then the reaction liquid containing the amplicons can be injected into the microfluidic pipeline from the reaction cavity to carry out hybridization color reaction. Therefore, the connection tube 110321 is not necessary, and in theory, the connection tube 110321 is not required as long as the connection hole on the side wall of the reaction chamber is closely attached to the liquid inlet on the microfluidic chip.
Specifically, referring to fig. 2a, 2b, and 2c, the smart mobile device includes a smart phone (i.e. the aforementioned smart terminal) and a smart phone adapter, the smart phone adapter mainly comprises an optical magnifier 210, an LED220, and a foldable paper bracket 230, the upper portion of the optical magnifier 210 is connected with a rear camera of the smart phone, and a 0.3W white LED220 is used as a light source module near the optical magnifier 210; the light source module may be powered by two button cells and operated by an on/off control switch, and the foldable paper holder 230 may be assembled from a black paper board.
Specifically, the foldable paper holder 230 has an insertion port 2301 and a detection signal intake port 2302, and when the nucleic acid detecting module is inserted into the foldable holder from the insertion port 2301 and a signal intake mechanism of a smart terminal is provided at the detection signal intake port 2302, the smart terminal can collect a detectable signal in the nucleic acid detecting module, wherein the optical magnifier 210 is provided at the detection signal intake port 2302.
The instant nucleic acid detection system provided by the embodiment of the invention can be applied to various nucleic acid detection methods, including but not limited to isothermal recombinase polymerase-gold-silver (iRPAS) triple signal amplification methods and the like, wherein the iRPAS method can realize exponential and visual signal amplification.
In a more specific embodiment, the method for detecting nucleic acid using the nucleic acid on-line detection device mainly comprises three main procedures: (i) sample pretreatment, (ii) signal amplification, (iii) signal readout to effect nucleic acid detection of the sample from the sample to the result.
Specifically, the invention adopts an isothermal recombinase polymerase-gold-silver (iRPAS) triple signal amplification method to amplify the collected DNA, wherein, firstly, a forward primer with tail and a biotinylation reverse primer are adopted to amplify the nucleic acid on an extraction test paper 11011 through recombinase polymerase, and the first signal amplification is realized by 20 minutes at 37 ℃; then, rotating rotary valve 1102 opens polytetrafluoroethylene connecting tube 110321, mix streptavidin-modified nano gold buffer solution and amplicon in connecting tube 110321 into detection assembly 120, and capture amplicon by base complementary reaction between pre-coated probe and amplicon tail in aldehyde-functionalized modified glass substrate (the binding point of probe and amplicon in the embodiment of the invention is located at tail, the binding point of amplicon and probe specificity can be located in middle and other areas), so that streptavidin-modified nano gold and biotin on amplicon are combined in microfluidic channel 1201, realizing second signal amplification, benefiting from high surface area and volume ratio of microfluidic channel, and fully obtaining signal in a very short reaction time; and, after washing with distilled water, the gold nano solution can nucleate silver deposition with high specificity by loading a mixture of silver salt and initiator solution into the microfluidic channel 1201 for further amplification and obtaining a visualized signal, and furthermore, silver deposited on the gold nano will cascade a catalytic effect to deposit more silver, thereby realizing a third signal amplification; finally, after the iRPAS triplex signal is amplified, the signal may be visually observed or semi-quantified by the smartphone, and in the signal readout step, the microfluidic detection assembly 120 is inserted into the bottom entry 2301 of the smartphone base, the signal on the microfluidic detection assembly 120 is captured by the camera, and the signal result will be read out and uploaded to the cloud database through the custom application.
Specifically, in order to absorb and enrich nucleic acid in a sample, a filter basket 1101 with a DNA extraction test paper 11011 built in the bottom is used to soak a natural sample such as several microliters of saliva or blood or urine or plant juice or sewage, cells are cracked and inactivated on the surface of the test paper, nucleic acid is stabilized on the DNA extraction test paper 11011 through electrostatic adsorption, after drying, the filter basket 1101 is inserted into a washing chamber 11031, and the reagent is stored in a polytetrafluoroethylene tube due to low consumption of the reagent, and a port is sealed by using an adhesive tape, so that the sample can be conveniently used, more importantly, different reagents can be separated through an air gap in one tube, and multiple reagents can be added in one tube.
Specifically, in the present embodiment, a purification reagent, TE buffer (10 mM Tris-HCl,0.1mM EDTA) and distilled water are sequentially injected into the washing chamber 11031 through the top port 11012 of the filter basket 1101; after purification, filter basket 1101 is inserted into reaction chamber 11032 to begin the signal amplification step.
Further, the bottom port of the filter basket 1101 in the embodiment of the present invention includes a hole or slit or the like capable of communicating with the outside, which is used to contact the sample with the DNA extraction reagent, the DNA amplification reagent or the like on the one hand, and to allow the washing reagent introduced into the filter basket 1101 to flow out when the filter basket 1101 is put into the washing chamber for washing and then taken out, at which time the amplicon (i.e., amplified DNA) is adsorbed on the DNA extraction test paper.
Specifically, in order to realize isothermal amplification at 37 ℃ and rapid heat transfer of the microfluidic detection assembly 120, the invention heats the smart phone and further provides a preset temperature condition for the whole iRPAS amplification process, and in order to realize stable heating of iRPAS amplification at 37 ℃, the invention provides a heating application (various application programs for extracting the temperature of the smart phone, such as a robusta APP, etc., which are known to those skilled in the art and are not described in detail herein), to read the data of the temperature sensor inside the smart phone, and regulate the heat of the smart phone by controlling the number of operation algorithms, the application program integrates temperature and time control functions, and the start and stop time of heating can be set by the application program.
According to the embodiment of the invention, the heat generated by mobile equipment such as a smart phone and the like is utilized, and the temperature of the surface of the smart phone or the temperature close to the smart phone is maintained or changed by installing a preset program on the smart phone, so that the detection chip is placed on the surface of the smart phone to be contacted with the smart phone or the smart phone and the detection chip are placed in a heat preservation container together, and further the iRPAS amplification process in the detection chip is maintained by the heat generated by the smart phone; the mobile phone control interface when the heating application or program is running is shown in fig. 9, and the control flow structure for controlling the number of running algorithms to adjust the heat of the smart phone is shown in fig. 10, where the algorithms and program codes for adjusting the heat of the smart phone and heating the reaction by using the heat can be implemented by using the prior art.
In order to simulate the effect of on-site test temperature conditions, the invention tested the performance of the detection platform (i.e., the nucleic acid instant detection system) at 4 ℃,25 ℃ and 37 ℃; the invention can reach the working temperature at 20min by adjusting the number of the operation algorithms, the precision reaches 0.1 ℃, and the result at 4 ℃ shows that the invention has comparable detection performance at 25 ℃ and 37 ℃. The instant nucleic acid detection system provided by the invention can be used in different temperature environments, and the temperature of the whole iRPAS amplification process is kept at the test temperature of 37-42 ℃ to ensure the detection of nucleic acid reaction.
As shown in fig. 8, the present invention provides an insulation container, such as an insulation bag made of aluminum foil, to reduce heat loss in cold environment (4 deg.c test), which is composed of a temperature display bar (30-40 deg.c, 2 deg.c interval) and a temperature quality control panel (37 deg.c, 42 deg.c), displaying real-time temperature inside, and recording whether the temperature reaches a working line and a dangerous line.
Specifically, the plasmid DNA was isolated from 10 11 The test was performed in the range of copy to 10 copies, and the lower limit of detection of the present invention was 113 copies per ml.
For example, a method for detecting nucleic acid using the nucleic acid on-the-fly detection system comprises:
1) Immersing the filter basket 1101 with the DNA extraction test paper 11011 arranged at the bottom thereof in a sample, and inserting the filter basket 1101 into the washing cavity 11031 after the sample is dried; then 200. Mu.l of a nucleic acid purification solution (FTA purification reagent from GE company in the United states) built in a polytetrafluoroethylene tube was injected into the washing chamber 11031 through the top port 11012 of the filter basket 1101 for 5 minutes, 200. Mu.l of TE buffer (containing 10mM Tris-HCl,0.1mM EDTA,pH 8.0) built in a polytetrafluoroethylene tube was then injected into the washing chamber 11031 for 5 minutes, and 200. Mu.l of distilled water was then injected for washing to complete the extraction and purification of DNA;
2) Inserting the filter basket 1101 with the DNA extracted into the reaction chamber 11032 to start the signal amplification step; specific:
2.1 To reaction chamber 11032 was added RPA reagents (available from twist dx corporation, england) including 25 μl of twist dx 2 x reaction buffer, 5 μl of 10 x base E mix, 3.6 μl of 25mM dNTP, 6.6 μl of water, 2.5 μl of core mix, then 4.8 μl of 10 uM biotin-modified DNA amplification primer, 2.5 μl of 280mM magnesium acetate solution, incubated at 37 ℃ for 20 minutes to effect first signal amplification;
2.2 After the reaction is completed, 50 μl of hybridization buffer solution containing 5×denhardt's solution, 3×sodium citrate solution, 0.1% bovine serum albumin and 1% avidin modified nano-gold particles is added into the reaction chamber 11032, the rotary valve 1102 is rotated to allow the reaction chamber 11032 to communicate with the connecting tube 110321, and the liquid in the reaction chamber is pushed into the microfluidic detection assembly 120 by injecting air into the reaction chamber 11032, and stored at 37 ℃ for 15min in the microfluidic channel, thereby realizing second signal amplification;
2.3 Distilled water was injected into the microfluidic detection module 120 to flush the channel, and 10 μl of silver staining solution and 10 μl of silver staining developing solution (all purchased from sigma corporation, usa) were injected into the microfluidic channel, and maintained at 37 ℃ for 7 minutes to achieve a third signal amplification.
3) And observing and analyzing the DNA amplified by the signal by adopting a smart phone.
Further, the instant detection system and method for nucleic acid provided in an exemplary embodiment of the present invention are used to detect a type α thalassemia SEA (long fragment deletion), a type β thalassemia CD41 type (short fragment deletion), a type β thalassemia CD17 type (single base substitution), a type β thalassemia CD71 type (single base insertion), an acetaldehyde dehydrogenase gene ALDH2 Single Nucleotide Polymorphism (SNP) wild type, an acetaldehyde dehydrogenase gene ALDH2 Single Nucleotide Polymorphism (SNP) mutation heterozygous type, and an acetaldehyde dehydrogenase gene ALDH2 Single Nucleotide Polymorphism (SNP) homozygous mutant type, and the detection results are shown in fig. 3a to 3d, respectively.
The instant nucleic acid detection device and method provided by the invention are used for detecting escherichia coli (E.coli) in a urine sample, klebsiella pneumoniae (K.pn) in a urine sample, escherichia coli (E.coli) in a milk sample, escherichia coli (E.coli) in a river sample and kiwi fruit canker (P.sa) in a kiwi fruit leaf sample, and detection results are respectively shown in figures 4 a-4 e.
Under different environmental temperature conditions, the intensity of a detection signal obtained by detecting nucleic acid by using the nucleic acid detection system provided by the embodiment of the invention is shown in fig. 5, wherein a correlation curve of the concentration of target DNA and the intensity of the detection signal is shown in fig. 6; the temperature effect of using the smart phone to heat the nucleic acid detection module under different ambient air temperature conditions is shown in fig. 7.
According to the nucleic acid instant detection system provided by the invention, the pretreatment component and the microfluidic component are combined, the iRPAS signal amplification method is adopted to amplify the extracted nucleic acid, and the intelligent mobile phone is used to heat the amplification process, so that the full integration of sample collection, nucleic acid release, purification, amplification and detection is realized.
Compared with the technical scheme provided in CN107904161A, the invention does not need to dilute the sample with normal saline and centrifuge centrifugation, does not need to use a magnetic bead method and a magnetic column for assistance, does not need to use a pipette for reagent sample adding, and does not need to provide heat by an additional constant temperature hot plate.
Compared with the technical scheme provided in CN103308502B, the device provided by the invention integrates a sample pretreatment system, and has low requirements on samples. No concentration and purification of the sample is required.
Compared with the technical scheme provided in US9469871B2, the device provided by the invention can fully exert the advantages of the microfluidic chip, and does not need the assistance of an external heating device.
It should be understood that the above embodiments are merely for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and implement the same according to the present invention without limiting the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (22)
1. A nucleic acid detection module characterized by comprising a preprocessing component (110) and a microfluidic detection component (120);
wherein the preprocessing component (110) comprises:
a nucleic acid extraction element for extracting at least nucleic acids in a sample, the nucleic acid extraction element comprising a filter basket (1101), a DNA extraction strip (11011) being provided within the filter basket (1101), the filter basket (1101) having a top port (11012) and a bottom port (11013), the top port (11012) being for injecting a wash reagent into the filter basket (1101), the bottom port (11013) being for letting the sample into the filter basket (1101) and into contact with the DNA extraction strip (11011), the wash reagent supplied by the wash reagent supply tube being able to enter the wash chamber (11031) through the inner chamber of the filter basket (1101) when the filter basket (1101) is locally inserted into the wash chamber (11031);
a main body module (1103) comprising a washing cavity (11031) and a reaction cavity (11032) which are matched with the nucleic acid extraction element, wherein the washing cavity (11031) is used for containing a washing reagent, a liquid discharge hole (110311) is arranged at the bottom of the washing cavity (11031), the washing reagent is used for purifying the nucleic acid, and the reaction cavity (11032) is used for containing a nucleic acid amplification reaction system capable of amplifying the nucleic acid to form an amplicon;
the microfluidic detection assembly (120) comprises a microfluidic channel and a temporary probe channel, the microfluidic channel is connected with the reaction cavity (11032), the microfluidic channel is communicated with the temporary probe channel, the temporary probe channel is at least used for providing a probe, the microfluidic channel is used for accommodating a detection reaction system containing the amplicon and the auxiliary detection reagent, the detection reaction system can generate a detectable signal, and the microfluidic channel and the temporary probe channel are respectively distributed in a microfluidic channel layer (1201) and a temporary probe channel layer (1202) of the microfluidic detection assembly (120).
2. The nucleic acid detection module of claim 1, wherein: the wash reagent supply pipe has an air gap therein to separate different wash reagents from each other.
3. The nucleic acid detection module of claim 1, wherein: when the filter basket (1101) is partially inserted into the reaction chamber (11032), the nucleic acid adsorbed on the DNA extraction test paper (11011) can be matched with other components required by the nucleic acid amplification reaction in the reaction chamber (11032) to form a nucleic acid amplification reaction system, so as to form amplicons.
4. The nucleic acid detection module of claim 1, wherein: the filter basket (1101) is provided with a connecting part (11014) matched with the valve (1102), the reaction cavity (11032) is provided with a connecting pipe (110321) communicated with the microfluidic detection assembly, when the filter basket (1101) is connected with the reaction cavity (11032) through the valve (1102), and the valve (1102) is in an open state, the inner cavity of the filter basket (1101) is communicated with the pipeline (110321) through a liquid outlet hole on the valve (1102).
5. The nucleic acid detection module of claim 4, wherein: the valve (1102) has an insertion hole (11024) and a discharge hole (11023), and when the filter basket (1101) is partially inserted into the insertion hole (11024), the inner cavity of the filter basket (1101) communicates with the discharge hole (11023).
6. The nucleic acid detection module of claim 4, wherein: the valve (1102) also has a groove (11021) for mounting a sealing ring.
7. The nucleic acid detection module of claim 4, wherein: the valve (1102) also has a rotational positioning index (11022), and the reaction chamber (11032) also has a positioning groove (110322) that mates with the rotational positioning index (11022) of the valve (1102).
8. The nucleic acid detection module of claim 1, wherein: at least one connecting protrusion (11033) and/or at least one connecting groove (11034) are arranged on one main body module, and the at least one connecting protrusion (11033) and the at least one connecting groove (11034) are respectively matched with the corresponding at least one connecting protrusion (11033) and the corresponding at least one connecting groove (11034) on the other main body module.
9. The nucleic acid detection module of claim 1, wherein: the auxiliary detection reagent comprises a nano-gold buffer solution with streptavidin modification, and the nucleic acid amplification reaction system comprises a tailed forward primer and a biotinylated reverse primer.
10. The nucleic acid detection module of claim 9, wherein: the auxiliary detection reagent also comprises a mixed solution of silver salt and an initiator.
11. The nucleic acid detection module of claim 9, wherein: the probe is complementary to an amplicon tail base derived from the tailed forward primer.
12. The nucleic acid detection module of claim 1, wherein: the preprocessing component (110) includes a 3D printing component.
13. The nucleic acid detection module of claim 1, wherein: the detection reaction system comprises an iRAS amplification detection reaction system.
14. The nucleic acid detection module of claim 1, wherein: the detectable signal comprises a visual detection signal.
15. A nucleic acid detecting unit characterized by comprising:
the nucleic acid detection module of any one of claims 1-14, and a smart terminal mated with the nucleic acid detection module, the smart terminal being configured to provide at least the heat required to facilitate a reaction for a nucleic acid amplification reaction system, a detection reaction system, performed within the nucleic acid detection unit.
16. The nucleic acid detection unit of claim 15, wherein: the intelligent terminal is also used for processing the detectable signal output by the nucleic acid detection module.
17. The nucleic acid detection unit of claim 15, wherein: the intelligent terminal comprises a mobile phone.
18. The nucleic acid detection unit of claim 15, further comprising a thermal container mated with the nucleic acid detection module, the thermal container having a temperature display strip and a temperature quality control disk.
19. A nucleic acid detection system, comprising:
the nucleic acid detection module of any one of claims 1-14 or the nucleic acid detection unit of any one of claims 15-18; and
the intelligent terminal adaptation module comprises a foldable support, wherein the foldable support is provided with an inserting port (2301) and a detection signal taking port (2302), when the nucleic acid detection module is inserted into the foldable support from the inserting port (2301), and a signal taking mechanism of the intelligent terminal is arranged at the detection signal taking port (2302), the intelligent terminal can collect a detectable signal in the nucleic acid detection module.
20. The nucleic acid detection system of claim 19, wherein: the foldable stand is a foldable paper stand (230).
21. The nucleic acid detection system of claim 19, wherein: the signal shooting mechanism comprises a camera, and an optical magnifier (210) matched with the camera is further arranged at the detection signal shooting port (2302).
22. The nucleic acid detection system of claim 19, wherein: the signal pickup mechanism comprises a camera, and a light source is arranged at the position corresponding to the detection signal pickup port (2302).
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