Nucleic acid detection module, detection unit and detection system
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 has important significance in life science, and is widely applied to clinical diagnosis, agricultural monitoring, food safety and detection of plant pathogen infection. Furthermore, with the intensive understanding of the human healthy genomic information, various types of nucleotide mutations such as gene substitutions, insertions and deletions have been proved to be closely related to diseases such as hereditary diseases, metabolic diseases and cancers, and even Single Nucleotide Polymorphisms (SNPs) have important correlations for individual disease susceptibility. Therefore, a method for detecting nucleic acid having high sensitivity and single base specificity is urgently required. Traditional nucleic acid detection methods such as DNA microarray, DNA sequencing, multiplex ligation dependent probe amplification, reverse dot blot, high resolution melting and some sensor based methods, etc., rely on expensive equipment and modern laboratory environments, preventing their application in on-site real-time nucleic acid detection in non-laboratory environments.
The instant detection is a detection means and trend which develops rapidly in recent years, namely a technical method for rapidly completing specimen collection, pretreatment, detection reaction and result reading and distinguishing on site. The method has the characteristics of rapidness, simplicity, convenience, high integration, no dependence on large-scale instruments and equipment, no dependence on professional workers and the like. The real-time detection provides possibility for the field rapid detection of food, water resource, environment and the like in underdeveloped areas and public health monitoring of basic units with limited resources, family health protection, disease prevention and the like. In recent years, many innovative detection methods have been developed for nucleic acid detection, and in particular, in isothermal nucleic acid amplification, for example, a loop-mediated isothermal amplification method, a rolling circle amplification method, a recombinase polymerase amplification method, etc. have been developed, and it is expected to be widely used for nucleic acid detection in the future.
The 3D printing microfluidic technology utilizes a new technology of directly printing a microfluidic chip or preparing a microfluidic chip mold by utilizing the 3D printing technology, and further promotes and extends the application range of microfluidic. Microfluidics (Microfluidics) is a technology appearing in the nineties of the last century, refers to the operation and control of fluids on a micrometer scale, has the characteristics of small volume, large specific surface area, small reagent dosage, high flux, high reaction speed and the like, and has a wide prospect in the field of instant detection. However, the preparation of the microfluidic chip depends on a semiconductor process, the processing process is various, and the preparation needs to be completed in an ultra-clean room, so that the application range of the microfluidic chip is limited. In recent years, 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 the visual point-of-care detection of pathogen nucleic acids is described in CN 107904161A. 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. It provides a micro-fluidic chip integrating sample treatment, pathogen nucleic acid extraction, amplification and visual detection. However, the operation of the chip still requires dilution of physiological saline of a sample, the centrifugal operation requires auxiliary operation by using magnetic beads and a liquid transfer device, and more importantly, the reaction still requires a constant-temperature 60-68 ℃ hot stage for heating, and the field instant detection cannot be completely realized. And, it adopts traditional photoetching micro-fluidic chip manufacturing process, and whole runner is the micron order, can't carry out liquid injection through manual for its easy and simple to handle nature greatly reduced, and the pretreatment of sample needs normal saline to dilute and centrifuge centrifugation just can concentrate the sample, adopts the magnetic bead method to need the magnetic column to assist, and the reagent application of sample need use the pipettor, and whole operation process is subject to the disadvantage of micro-fluidic chip: the manual operation is not easy.
In CN103308502B, a palm-type universal micro-fluidic chip real-time detection device and application are provided, which mainly adopt a fluorescence loop-mediated isothermal amplification method to amplify and detect nucleic acid, and the device integrates a mobile power supply heating system and a fluorescence detection system, overcomes the defect that the traditional nucleic acid detection reaction needs external power supply and is externally connected with a large-scale computer, greatly expands the applicable range of the device, is matched with an application program (APP) running on intelligent mobile equipment, is easy to operate, and can be applied to the nucleic acid amplification reaction; however, the scheme does not have a sample pretreatment system, the detection sample is a sample which is enriched and purified, the assistance of a nucleic acid enrichment and purification system is required, and the field instant detection cannot be completely realized; the instant nucleic acid detection device adopts a handheld microfluidic chip and adopts a fluorescence loop-mediated isothermal amplification method to carry out amplification detection on nucleic acid, and the device integrates a mobile power supply heating system and a fluorescence detection system; however, a sample pretreatment system is not provided, a detection sample is a concentrated and purified sample, the real-time on-site detection cannot be really realized, and the specificity of the method cannot reach the single base difference level.
A method and apparatus for on-the-fly nucleic acid amplification and detection is disclosed in US9469871B2, embodiments of which include a fully integrated sample-to-result molecular diagnostic instrument that preferably utilizes a loop-mediated isothermal amplification method to reduce instrument requirements associated with nucleic acid amplification and to enable detection of target amplification by color 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; and the method is mainly based on purification and detection of nucleic acid on a macroscopic scale by using an injector and matched devices, a loop-mediated isothermal amplification method is also adopted, 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, so as to overcome the defects in the prior art.
In order to achieve the purpose, 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 pretreatment component (110) and a microfluidic detection component (120);
wherein the pre-processing assembly (110) comprises:
a nucleic acid extraction element for extracting at least nucleic acid in a sample;
a main body module (1103) comprising a washing chamber (11031) and a reaction chamber (11032) which are matched with the nucleic acid extraction element, wherein the washing chamber (11031) is used for accommodating washing reagent which is used for purifying the nucleic acid, and the reaction chamber (11032) is used for accommodating a nucleic acid amplification reaction system which can amplify the nucleic acid to form an amplicon;
the microfluidic detection assembly (120) comprises a microfluidic channel, the microfluidic channel is connected with a reaction cavity (11032), the microfluidic channel is used for accommodating a detection reaction system containing the amplicon and an auxiliary detection reagent, and the detection reaction system can generate 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 to be in contact with the DNA extraction test paper (11011).
Furthermore, when the filter basket (1101) is partially inserted into the washing chamber (11031), the washing reagent supplied by the washing reagent supply pipe can enter the washing chamber (11031) through the inner cavity 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 drain hole (110311).
Further, when the filter basket (1101) is partially inserted into the reaction cavity (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 cavity (11032) to form a nucleic acid amplification reaction system, and then an amplicon is 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, the filter basket (1101) is connected with the reaction cavity (11032) through the valve (1102), and when the valve (1102) is in an open state, the inner cavity of the filter basket (1101) is communicated with the pipeline (110321) through an outlet hole in the valve (1102).
Further, the valve (1102) is provided with an insertion hole (11024) and an outlet hole (11023), and when the filter basket (1101) is partially inserted into the insertion hole (11024), the inner cavity of the filter basket (1101) is communicated with the outlet 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 chamber (11032) is also provided with a positioning groove (110322) matched with the rotary positioning mark (11022) of the valve (1102)
Furthermore, 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.
Furthermore, the auxiliary detection reagent comprises a nano gold buffer solution modified by streptavidin, 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 a silver salt and an initiator.
In some more specific embodiments, the microfluidic detection assembly (120) further comprises a temporary probe channel, the microfluidic channel in communication with the temporary probe channel, the temporary probe channel at least for providing 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 pre-processing component (110) comprises 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 promoting the reaction for the nucleic acid amplification reaction system and the detection reaction system which are carried out in the nucleic acid detection unit.
Furthermore, a heating module is arranged in the intelligent terminal.
Further, the intelligent terminal comprises a mobile phone.
In some more specific embodiments, the nucleic acid detecting unit further comprises a heat-insulating container matched with the nucleic acid detecting module, and the heat-insulating container is provided with a temperature display strip and a temperature quality control panel.
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
a smart terminal adaptor module comprising a foldable stand having an insertion port (2301) and a detection signal intake port (2302), the smart terminal being capable of acquiring a detectable signal within the nucleic acid detection module when the nucleic acid detection module is inserted into the foldable stand from the insertion port (2301) and a signal intake mechanism of the smart terminal is disposed at the detection signal intake port (2302).
Further, the foldable support is a foldable paper support (230).
Further, 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).
Furthermore, the signal shooting mechanism comprises a camera, and a light source is arranged at a position corresponding to the detection signal shooting port (2302).
Compared with the prior art, the system for detecting the nucleic acid in real time combines the pretreatment component and the microfluidic component, amplifies the extracted nucleic acid by adopting an iRPAS signal amplification method, and heats the amplification process by using a smart phone, so that the full integration of sample collection, nucleic acid release, purification, amplification and detection is realized.
Drawings
FIG. 1a is a schematic diagram of a nucleic acid detection 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 diagram of a rotary valve in an exemplary embodiment of the present invention;
FIG. 2a is a schematic view of a folded paper holder according to an exemplary embodiment of the present invention
FIG. 2b is a schematic illustration of a disassembled foldable paper holder according to an exemplary embodiment of the present invention;
FIG. 2c is a schematic view of a folding process of a foldable stand according to an exemplary embodiment of the present invention, wherein the broken lines are folding lines;
FIG. 3a is the result of detection of α thalassemia SEA type (long fragment missing) using the instant nucleic acid detection system provided by the present invention;
FIG. 3b is the result of detection of β thalassemia CD41 (short fragment deletion) by the instant nucleic acid detection system;
FIG. 3c shows the detection result of β thalassemia CD17 (single base substitution) by using the instant nucleic acid detection system;
FIG. 3d is a diagram showing the results of detection of β thalassemia CD71 type (single base insertion) using the instant nucleic acid detection system provided by the present invention;
fig. 4a is a detection result of detecting escherichia coli (e.coli) in a urine sample by using the instant nucleic acid detection system provided by the present invention;
FIG. 4b is a diagram showing the detection result of Klebsiella pneumoniae (K.pn) in a urine sample by using the instant nucleic acid detection system provided by the present invention;
fig. 4c is a detection result of detecting escherichia coli (e.coli) in a milk sample by using the instant nucleic acid detection system provided by the present invention;
fig. 4d is a detection result of detecting escherichia coli (e.coli) in a river water sample by using the instant nucleic acid detection system provided by the present invention;
fig. 4e is a detection result of kiwi canker pathogen (p.sa) in kiwi fruit leaf sample by using the instant nucleic acid detection system provided by the present invention;
FIG. 5 is a graph showing the detection signal intensities obtained by detecting nucleic acids using the nucleic acid detecting 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 detection signal in an exemplary embodiment of the present invention;
FIG. 7 is a diagram showing the effect of heating the nucleic acid detecting module by the smart phone under different ambient temperature conditions;
FIG. 8 is a schematic diagram of an insulated bag according to an exemplary embodiment of the present invention;
fig. 9 is a diagram illustrating a mobile phone operating interface for operating a heating application or program in a smart phone according to an exemplary embodiment of the present invention;
fig. 10 is a block diagram of a control flow for controlling the number of operating algorithms to regulate the amount of heat in a smart phone in an exemplary embodiment of the invention.
Detailed Description
In view of the deficiencies in the prior art, the inventors of the present invention have made extensive studies and extensive practices to provide technical solutions of the present invention. The technical solution, its implementation and principles, etc. will be further explained as follows.
Referring to fig. 1 a-1 d, an instant nucleic acid detection system according to an exemplary embodiment of the present invention mainly comprises a detection chip (i.e., the aforementioned nucleic acid detection module, the same below) and a customized smart mobile device 200, which can be placed in a pocket by an end user and quickly assembled to be used in a resource-limited situation.
Referring to fig. 1 a-1 d, the detection chip mainly includes a pretreatment module 110 and a microfluidic detection module 120; the pre-processing assembly 110 is 3D printed and comprises a filter basket 1101 for sample absorption and DNA purification, a rotary valve (i.e. the aforementioned valve, the same below) 1102 for controlling the opening and closing of the microfluidic channels, and a body module 1103 for constructing the chamber 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 sealing ring placing groove 11021 is provided on the outer side surface of the rotary valve 1102, and a rotary positioning mark 11022 and a sidewall liquid outlet 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 connection tube (110321, the bottom is provided with a positioning groove 110322 for positioning the rotary valve, besides, one main body module is provided with at least one connection protrusion 11033 and/or at least one connection groove 11034, the at least one connection protrusion 11033 and the at least one connection groove 11031 are respectively matched with at least one corresponding connection protrusion and at least one corresponding connection 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 modified glass substrate, the microfluidic channels are distributed in the microfluidic channel layer 1201, and DNA or RNA probes capable of complementarily binding with the local sequences of the amplicons are hung on the surface of the probe region of the aldehyde-functionalized modified glass substrate.
Specifically, the temporary probe channel layer 1202 and the aldehyde-group functionalized glass substrate are combined through an aldehyde-amine condensation reaction, a detection probe is injected, the temporary probe channel layer 1202 is peeled off after the probe is coated on the surface of the aldehyde-group functionalized glass substrate, and the microfluidic channel layer 1201 and the aldehyde-group functionalized glass substrate are combined through the 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 regions of the microfluidic channel; in the embodiment of the invention, different nucleic acid probes can be fixed in different areas of the microfluidic channel in advance; for example, the probes may be immobilized at specific locations of the microfluidic channel by printing, or the like, or temporary probe channels may be placed at different regions of the microfluidic channel to immobilize different nucleic acid probes.
Specifically, the rotary valve 1102 in this embodiment is a sleeve with an outlet hole on the sidewall, and the filter basket 1101 is inserted into the rotary valve (sleeve) 1102 and then inserted into the reaction chamber 11032; because the liquid outlet holes on the side wall of the rotary valve 1102 are staggered with the holes on the side wall of the reaction chamber 11032, the reaction liquid can be remained in the reaction chamber 11032, but the rotary valve 1102 is not necessary, and the filter basket 1101 can also 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 the connecting holes and the external connecting pipe 110321 to be 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 very small, the liquid needs a certain pressure as a thrust when entering the microfluidic pipeline; the rotary valve 1102 is used only to prevent the reaction solution from leaking into the microfluidic channel in advance during the reaction.
Specifically, the connection pipe 110321 is communicated with the reaction chamber through a connection hole on the side wall of the reaction chamber, when the liquid outlet hole 11023 is dislocated with the connection hole, the solution in the filter basket 1101 is not communicated with the connection pipe 110321, the solution in the reaction chamber 11032 does not flow outwards, and the reaction in the reaction chamber 11032 is carried out; after the reaction is finished, the whole rotary valve 1102 is rotated to enable the liquid outlet holes 11023 to be opposite to the connecting holes, the reaction cavity 11032 is communicated with the connecting pipe 110321, air is pushed from the valve 11012, and then the reaction liquid containing the amplicon can be injected into the microfluidic pipeline from the reaction cavity to carry out hybridization color development reaction. Therefore, the connection pipe 110321 is not necessary, and theoretically, the connection pipe 110321 is not necessary 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, please refer to fig. 2a, fig. 2b, and fig. 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 includes an optical magnifier 210, an LED220, and a foldable paper holder 230, an upper portion of the optical magnifier 210 is connected to a rear camera of the smart phone, and a 0.3W white LED220 is used near the optical magnifier 210 as a light source module; 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 black cardboard.
Specifically, the foldable paper holder 230 has an insertion opening 2301 and a detection signal intake opening 2302, and when the nucleic acid detection module is inserted into the foldable holder from the insertion opening 2301 and the signal intake mechanism of the smart terminal is disposed at the detection signal intake opening 2302, the smart terminal can collect a detectable signal in the nucleic acid detection module, wherein the optical magnifier 210 is disposed at the detection signal intake opening 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 an isothermal recombinase polymerase-gold-silver (iRPAS) triple signal amplification method 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 by using the instant nucleic acid detection device mainly comprises three main procedures: (i) sample pre-treatment, (ii) signal amplification, (iii) signal readout to enable nucleic acid detection of the sample from sample to result.
Specifically, the collected DNA is amplified by adopting an isothermal recombinase polymerase-gold-silver (iRPAS) triple signal amplification method, wherein a tailed forward primer and a biotinylated reverse primer are adopted to amplify and extract nucleic acid on test paper 11011 through recombinase polymerase, and the first signal amplification is realized at 37 ℃ for 20 minutes; then, the rotary valve 1102 is rotated to open the polytetrafluoroethylene connecting pipe 110321, the streptavidin modified nanogold buffer solution in the connecting pipe 110321 and the amplicon are mixed and injected into the detection assembly 120, and base complementary reaction is performed between the probe pre-coated in the aldehyde group functionalized modified glass substrate and the tail of the amplicon (the binding point of the probe and the amplicon in the embodiment of the invention is located at the tail, and the specific binding point of the amplicon and the probe can also be located in other areas such as the middle), so that the amplicon is captured, the streptavidin modified nanogold and the biotin on the amplicon are bound in the microfluidic channel 1201, secondary signal amplification is realized, signals are fully obtained in a short reaction time due to the high surface area to volume ratio of the microfluidic channel; and, after washing with distilled water, in order to further amplify and obtain a visualized signal, the nanogold solution can nucleate the deposition of silver with high specificity by loading a mixture of silver salt and initiator solution into the microfluidic channel 1201 for reaction, and in addition, the silver deposited on the nanogold will cascade catalytic effect to deposit more silver, thereby realizing a third signal amplification; finally, after the iRPAS triple signal is amplified, the signal can be observed by naked eyes or semi-quantified by a smart phone, in the signal reading step, the microfluidic detection assembly 120 is inserted into the bottom inlet 2301 of the smart phone base, the signal on the microfluidic detection assembly 120 is captured by a camera, and the signal result is read out and uploaded to a cloud database through a customized application program.
Specifically, in order to absorb and enrich nucleic acids in a sample during sample treatment, the filter basket 1101 with the bottom provided with the DNA extraction test paper 11011 is used for soaking a few microliters of natural samples such as saliva, blood, urine, plant juice or sewage, cells are cracked and inactivated on the surface of the test paper, the nucleic acids are stabilized on the DNA extraction test paper 11011 through electrostatic adsorption, and after drying, the filter basket 1101 is inserted into the washing cavity 11031.
Specifically, in the embodiment of the present invention, the purification reagent, TE buffer (10mM Tris-HCl, 0.1mM EDTA) and distilled water are injected into the wash chamber 11031 sequentially through the top port 11012 of the filter basket 1101; after purification, the filter basket 1101 is inserted into the 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 holes or slits capable of communicating with the outside, and the bottom port is used to contact the sample with the DNA extraction reagent, the DNA amplification reagent, and the like, and to allow the washing reagent entering 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 temperature by using an intelligent mobile phone, and further provides a preset temperature condition for the whole iRPAS amplification process, 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 mobile phone, such as ludaoshi APP and the like, the technology is the prior art known by the technical personnel in the field, and is not described herein any more) for reading the data of the temperature sensor inside the intelligent mobile phone and adjusting the heat of the intelligent mobile phone by controlling the number of operation algorithms, the application program integrates the temperature and time control function, and the starting and stopping 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, the surface temperature 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, a detection chip is placed on the surface of the smart phone to be in contact with the smart phone or the smart phone and the detection chip are placed in a heat-preserving container together, and 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 algorithm and program code 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 influence of field test temperature conditions, the invention tests the performance of a detection platform (namely the nucleic acid instant detection system) at 4 ℃, 25 ℃ and 37 ℃; according to the invention, the temperature is adjusted to reach the working temperature within 20min by adjusting the number of the operation algorithms, the precision reaches 0.1 ℃, and the result at 4 ℃ shows that the detection performance is comparable 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 ℃ so as to ensure the detection of nucleic acid reaction.
As shown in fig. 8, the present invention provides an insulated container, such as an insulated bag made of aluminum foil, to reduce heat loss in cold environment (4 c test), which is composed of temperature display bars (30-40 c, 2c interval) and a temperature quality control panel (37 c, 42 c), to display the real-time temperature inside, and to record whether the temperature reaches the working line and the hazard line.
Specifically, by subjecting plasmid DNA to DNA extraction from 1011The copy is tested in the range of 10 copies, and the lower limit of detection in the present invention is 113 copies per ml.
For example, a method for detecting nucleic acid using the instant nucleic acid detection system includes:
1) dipping a sample in a filter basket 1101 with DNA extraction test paper 11011 arranged at the bottom, and inserting the filter basket 1101 into a washing cavity 11031 after drying; then injecting 200 microliters of nucleic acid purification solution (purchased from FTA purification reagent of GE company in USA) in the polytetrafluoroethylene tube into the washing cavity 11031 through the top port 11012 of the filter basket 1101, keeping for 5 minutes, then injecting 200 microliters of TE buffer solution (containing 10mM Tris-HCl, 0.1mM EDTA, pH 8.0) in the polytetrafluoroethylene tube into the washing cavity 11031, keeping for 5 minutes, and then injecting 200 microliters of distilled water for cleaning, so as to complete the extraction and purification of DNA;
2) inserting the filter basket 1101 with the extracted DNA into the reaction chamber 11032 to start a signal amplification step; specifically, the method comprises the following steps:
2.1) adding RPA reaction reagent (purchased from TwistDx Co., UK) including 25. mu.L of LTwistdx 2 Xreaction buffer, 5. mu.L of 10 Xbasic E mixture, 3.6. mu.L of 25mM dNTP, 6.6. mu.L of water, 2.5. mu.L of core mixture to the reaction chamber 11032, then adding 4.8. mu.L of 10 uM biotin-modified DNA amplification primer, 2.5. mu.L of 280mM magnesium acetate solution, and incubating at 37 ℃ for 20 minutes to effect first signal amplification;
2.2) after the reaction, adding 50 μ L 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 into the reaction cavity 11032, rotating the rotary valve 1102 to communicate the reaction cavity 11032 with the connecting pipe 110321, injecting air into the reaction cavity 11032, pushing the liquid in the reaction cavity into the microfluidic detection assembly 120, storing for 15min at 37 ℃ in the microfluidic channel, and realizing the second signal amplification;
2.3) injecting distilled water into a washing channel of the microfluidic detection assembly 120, then injecting 10 mu L of silver staining solution and 10 mu L of silver staining developing solution (both purchased from sigma company of America) into the microfluidic channel, and keeping the temperature at 37 ℃ for 7 minutes to realize third signal amplification.
3) And observing and analyzing the amplified DNA by using a smart phone.
Further, the instant nucleic acid detection system and method provided by an exemplary embodiment of the present invention are used to detect α thalassemia SEA type (long fragment deletion), β thalassemia CD41 type (short fragment deletion), β thalassemia CD17 type (single base substitution), β thalassemia CD71 type (single base insertion), acetaldehyde dehydrogenase gene ALDH2 Single Nucleotide Polymorphism (SNP) wild type, acetaldehyde dehydrogenase gene ALDH2 Single Nucleotide Polymorphism (SNP) mutation heterozygous type, and acetaldehyde dehydrogenase gene ALDH2 Single Nucleotide Polymorphism (SNP) homozygous mutant type, and the detection results are shown in fig. 3a to fig. 3g, respectively.
The device and the method for detecting the nucleic acid in real time are adopted to detect escherichia coli (E.coli) in a urine sample, klebsiella pneumoniae (K.pn) in the urine sample, escherichia coli (E.coli) in a milk sample, escherichia coli (E.coli) in a river water sample and kiwifruit canker pathogen (P.sa) in a kiwi fruit leaf sample, and the detection results are respectively shown in figures 4 a-4 e.
Under different environmental temperature conditions, the detection signal intensity 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 the correlation curve of the target DNA concentration and the detection signal intensity is shown in FIG. 6; under different ambient temperature conditions, the temperature effect of heating the nucleic acid detection module by the smart phone is shown in fig. 7.
The instant nucleic acid detection system provided by the invention combines the pretreatment component and the microfluidic component, amplifies the extracted nucleic acid by adopting an iRPAS signal amplification method, and heats the amplification process by using a smart phone, thereby realizing the fully integrated integration of sample collection, nucleic acid release, purification, amplification and detection.
Compared with the technical scheme provided in CN107904161A, the method provided by the invention does not need to dilute the sample with physiological saline and centrifuge, does not need to adopt a magnetic bead method to assist with a magnetic column, does not need to use a pipettor for reagent sample adding, and does not need an additional constant temperature hot plate to provide heat.
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. The sample does not need to be concentrated and purified.
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-mentioned embodiments are merely illustrative of the technical concepts and features of the present invention, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and therefore, the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.