CN113376126B - Portable loop-mediated isothermal amplification device and operation method thereof - Google Patents
Portable loop-mediated isothermal amplification device and operation method thereof Download PDFInfo
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Abstract
The invention relates to a portable loop-mediated isothermal amplification device and an operation method thereof, wherein the portable loop-mediated isothermal amplification device comprises: the device comprises a constant temperature heating module, an excitation and detection module, a shell module, a control module and an output module; after the reaction of the sample is terminated, the control module analyzes the reaction rule of the sample according to photoelectric signal data received at fixed time intervals, and performs outlier rejection and weighting operation on the photoelectric signal data; the invention is portable, has low cost and no special environmental requirement, and is suitable for household detection; the fully-enclosed constant temperature and heat preservation design is adopted, so that the liquid is prevented from evaporating to the pipe wall and the upper cover during the reaction of the micro-sample, and the reaction precision is prevented from being influenced; the closed light path design is adopted, the indicating lamp can output the interpretation result in the reaction process in real time, human eyes are replaced for interpretation, and the reading error is reduced.
Description
Technical Field
The invention belongs to the technical field of loop-mediated isothermal amplification, in particular to a portable loop-mediated isothermal amplification device and an operation method thereof, which can read reaction process data and judge reaction results in real time.
Background
Currently, loop-mediated isothermal amplification technology is widely used in detection of SARS, avian influenza, HIV, covid-9 and other diseases. The loop-mediated isothermal amplification has the advantages of high specificity, high sensitivity, simple operation and the like, the reaction can be realized without a PCR instrument and expensive reagents, the water bath or the metal bath, and the reaction result is judged by naked eye observation;
however, more and more scientific experiments find that due to the influence of factors such as different concentrations of the selected primers, unstable reaction temperature and the like, the mode of judging the reaction result by visual observation after the reaction is finished has higher misjudgment rate, the reaction phenomenon in the reaction process cannot be obtained in real time, and the detection requirement with higher requirements cannot be met. It is necessary to develop a loop-mediated isothermal amplification device capable of reading reaction process data in real time and judging reaction results.
Disclosure of Invention
In order to overcome the technical defects, the invention provides a portable loop-mediated isothermal amplification device and an operation method thereof, and aims to provide a portable isothermal amplification instrument and a detection method thereof, wherein a multi-sample multi-channel optical detection system can be carried, the reaction phenomenon can be read in real time, and the miniaturization of the isothermal amplification instrument can be realized.
A portable loop-mediated isothermal amplification device and an operating method thereof, wherein:
a portable loop-mediated isothermal amplification device comprising: the device comprises a constant temperature heating module, an excitation and detection module, a shell module, a control module and an output module;
further, the isothermal heating module is used for providing isothermal environment required by sample reaction, and comprises: the device comprises an upper heat cover, a sample constant temperature tank and a base plate; the upper heat cover is integrated with a first heating material, a first temperature sensor and a first temperature protection switch; an upper heat cover constant temperature protection layer is surrounded on the top and the periphery of the upper heat cover; the sample constant temperature tank is integrated with a second heating material, a second temperature sensor, a second temperature protection switch and the like; the sample constant temperature tank is arranged in the substrate; the bottoms and the peripheries of the sample groove and the substrate are wrapped by a sample constant temperature groove protection layer; during reaction, the reaction system is arranged in a sample tube, and the sample tube is arranged in a constant temperature space between the upper heat cover and the sample constant temperature tank; the first heating material, the first temperature sensor and the first temperature protection switch are electrically connected with the control module;
as an example, the first heat generating material and the second heat generating material are: one or a combination of heating films or heating sheets;
as an illustration, the sample tube includes, but is not limited to, a PCR tube or a microfluidic chamber;
further, the excitation and detection module includes: an excitation module and a detection module;
the excitation module is used for exciting a luminous group in a sample system and comprises a plurality of excitation subsystems;
the excitation subsystem includes: an adjustable excitation light source, an adjustable excitation light source focusing lens; the adjustable excitation light source focusing lens is arranged in front of the adjustable excitation light source, and excitation light irradiates the sample tube through an incident light hole on the sample placing groove;
the detection module is used for receiving the light signals excited by the sample and comprises a plurality of detection subsystems; the detection module is arranged at the detection light receiving hole;
the detection subsystem includes: the optical filter, the detection focusing lens and the photoelectric sensor structure;
furthermore, the detection subsystems and the excitation subsystems corresponding to the same sample constant temperature tank are equal in number, symmetrical left and right and located at the same liquid level;
further, the excitation and detection module is arranged in the substrate, the sample constant temperature tank is provided with an incident light hole and a detection light receiving hole, and the incident light hole and the detection light receiving hole are symmetrically arranged at two ends of the sample constant temperature tank;
further, the shell module is used for bearing the constant-temperature heating module, the excitation and detection module, the control module and the output module;
the housing module includes: the upper cover, the hinge, the shell, the lock catch and the switch button; the upper cover is connected with the shell through a hinge, and can be opened and closed at 0-90 degrees relative to the shell by taking the hinge as a center, and the lock catch is triggered when the upper cover is closed, so that the upper cover and the shell are locked;
further, the upper heat cover, the first heating material, the first temperature sensor, the first temperature protection switch and the upper heat cover constant temperature protection layer are arranged in the upper cover; the sample constant temperature tank, the second heating material, the second temperature sensor, the second temperature protection switch, the substrate and the sample constant temperature tank protection layer are arranged in the shell;
further, the control module is used for adjusting the temperature of the constant-temperature heating module, controlling the excitation and detection module to work, collecting signals of the detection module in real time and judging and reading sample properties;
further, the output module is arranged at the front end of the shell and is used for displaying the interpretation result so as to enable a user to identify the detection result.
A method of operating a portable loop-mediated isothermal amplification device, comprising:
step one, a control module starts a constant temperature heating module to start heating, and adjusts the temperature according to a feedback value of a temperature sensor so that the reaction temperature of a sample is kept under a constant temperature condition;
as an illustration, the constant temperature conditions are: 65 ℃;
as an illustration, according to the LAMP reaction mechanism, the requirements for the temperature environment at the time of LAMP reaction are generally: about 65 degrees;
step two, the control module controls the excitation module to irradiate excitation light on the sample;
step three, the control module controls the detection module to receive the optical signal generated after the sample is excited, and then the optical signal is subjected to photoelectric conversion and processing to form a photoelectric signal and then transmitted to the control module;
step four, the control module presets the total reaction time, and receives photoelectric signal data according to fixed time intervals in the reaction time;
as an illustration, the photoelectric signal data is reaction data;
step five, after the reaction of the sample is terminated, the control module analyzes the reaction rule of the sample according to the photoelectric signal data received at fixed time intervals, and performs outlier rejection and weighting operation on the photoelectric signal data;
as an example, assuming that the total number of photoelectric signals received by the control module is N from the start of the reaction to the end of the reaction, the signal value obtained at each sampling point is D [i] (0.ltoreq.i.ltoreq.N), (i representing one of N sampling points);
as an illustration, outlier rejection refers to: the total reaction time was set as: 30 minutes, 2 times per minute, each sampling data is X i (i represents one of N sampling points, i is 0.ltoreq.i)<N, in this example n=60), is taken
When |X i -X mean When the I is more than 3 sigma, the abnormal value is removed, and the average value of the data before and after the abnormal value is used for replacing the abnormal value;
as an illustration, the weighted operation refers to: since the reaction data varies in the degree of 30 minutes, the reaction data in 20 minutes to 30 minutes has more remarkable influence on the numerical result, the data after 10 minutes of reaction is weighted, the collected data of 10 minutes to 20 minutes is multiplied by 40% of weight, the data of 20 minutes to 30 minutes is multiplied by 60% of weight, and the weighted data is summed, that is
Step six, when the numerical result is within the range of the positive threshold value after the operation in the step five, the control module controls the output module to set the positive indicator lamp; when the numerical result is within the negative threshold range, the control module controls the output module to set a negative indicator lamp, so that a detection conclusion is provided for a user;
as an illustration, the output module is further configured to output the real-time reaction phenomenon of the visualized sample system and the sample attribute interpretation result;
the invention has the beneficial effects that:
1. the equipment provided by the invention is small and exquisite, portable, low in cost, free from special environmental requirements and suitable for household detection;
2. the invention adopts the fully enclosed constant temperature and heat preservation design, so as to prevent liquid from evaporating to the pipe wall and the upper cover during the reaction of the micro sample, and influence the reaction precision;
3. the invention adopts a closed light path design, the indicator lamp can output the interpretation result in the reaction process in real time, replaces human eye interpretation, and reduces errors caused by human eye interpretation;
drawings
FIG. 1 is a schematic diagram showing the internal structure of a portable loop-mediated isothermal amplification device according to the present invention
FIG. 2 is a schematic diagram showing the external structure of a portable loop-mediated isothermal amplification device according to the present invention
FIG. 3 is a schematic diagram of a portable loop-mediated isothermal amplification device according to the present invention, showing sample ratios without filters
FIG. 4 is a sample-to-optical schematic diagram of a portable loop-mediated isothermal amplification device incorporating a filter according to the present invention
FIG. 5 is a diagram showing an example of application of a portable loop-mediated isothermal amplification device according to the present invention
Detailed Description
The preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
As shown in fig. 1 to 5, a portable loop-mediated isothermal amplification device comprises: the constant temperature heating module, the excitation and detection module, the shell module, the control module and the output module 23;
further, the isothermal heating module is used for providing isothermal environment required by sample reaction, and comprises: an upper heat cover 1, a sample constant temperature tank 6 and a base plate 10; the upper heat cover 1 is integrated with a first heating material 2, a first temperature sensor 3 and a first temperature protection switch 4; the top and the periphery of the upper heat cover are surrounded by an upper heat cover constant temperature protection layer 5; the sample constant temperature tank 6 is integrated with a second heating material 7, a second temperature sensor 8, a second temperature protection switch 9 and the like; the sample constant temperature tank 6 is arranged inside the substrate 10; the bottoms and the peripheries of the sample groove 6 and the substrate 10 are wrapped by a sample constant temperature groove protection layer 11; in the reaction, a reaction system 18 is arranged in a sample tube 17, and the sample tube 17 is arranged in a constant temperature space between the upper heat cover 1 and the sample constant temperature tank 6; the first heating material 2, the first temperature sensor 3, the first temperature protection switch 4, the second heating material 7, the second temperature sensor 8 and the second temperature protection switch 9 are electrically connected with the control module;
as an example, the first heat generating material 2 and the second heat generating material 7 are: one or a combination of heating films or heating sheets;
as an example, the sample tube 17 includes, but is not limited to, a PCR tube or a reaction system such as a microfluidic chamber;
further, the excitation and detection module includes: an excitation module and a detection module; the excitation and detection module is arranged in the substrate 10, the sample constant temperature tank 6 is provided with an incident light hole and a detection light receiving hole, and the incident light hole and the detection light receiving hole are symmetrically arranged at two ends of the sample constant temperature tank 6;
the excitation module is used for exciting a luminous group in a sample system and comprises a plurality of excitation subsystems;
as an illustration, the excitation subsystem comprises: an adjustable excitation light source 12, an adjustable excitation light source focusing lens 13; the adjustable excitation light source focusing lens 13 is arranged in front of the adjustable excitation light source 12, and excitation light irradiates the sample tube through an incident light hole on the sample placing groove;
further, the detection module is used for receiving the optical signal of the excited sample, and comprises a plurality of detection subsystems; the detection module is arranged at the detection light receiving hole;
as an illustration, the detection subsystem is constituted by a filter 14, a detection focusing lens 15, a photosensor structure 16;
furthermore, the detection subsystems and the excitation subsystems corresponding to the same sample constant temperature tank 6 are equal in number, symmetrical left and right and located at the same liquid level;
further, the housing module includes: upper cover 19, hinge 20, housing 21, latch 22, and switch button 24; the upper cover 19 is connected with the shell 21 by a hinge 20, and can be opened and closed at 0-90 degrees relative to the shell by taking the hinge as a center, and the lock catch 22 is triggered when the upper cover is closed, so that the upper cover and the shell are locked.
Further, the upper heat cover 1, the first heating material 2, the first temperature sensor 3, the first temperature protection switch 4 and the upper heat cover constant temperature protection layer 5 are arranged inside the upper cover 19; the sample constant temperature tank 6, the second heating material 7, the second temperature sensor 8, the second temperature protection switch 9, the substrate 10 and the sample constant temperature tank protection layer 11 are arranged in the shell 21;
further, the control module is used for adjusting the temperature of the constant-temperature heating module, controlling the excitation and detection module to work, collecting signals of the detection module in real time and judging and reading sample properties;
further, the output module 23 is disposed at the front end of the housing 21, and is used for displaying the interpretation result, so as to allow the user to identify the detection result.
A method of operating a portable loop-mediated isothermal amplification device, comprising:
step one, a control module starts a constant temperature heating module 1 to start heating, and adjusts the temperature according to the feedback value of a temperature sensor 21 so that the reaction temperature of a sample is kept under a constant temperature condition;
as an illustration, the constant temperature conditions are: 65 ℃;
as an illustration, according to the LAMP reaction mechanism, the requirements for the temperature environment at the time of LAMP reaction are generally: about 65 degrees;
step two, the control module controls the excitation module to irradiate excitation light on the sample;
step three, the control module controls the detection module to receive the optical signal generated after the sample is excited, and then the optical signal is subjected to photoelectric conversion and processing to form a photoelectric signal and then transmitted to the control module;
step four, the control module presets the total reaction time, and receives photoelectric signal data according to fixed time intervals in the reaction time;
as an illustration, the photoelectric signal data is reaction data;
step five, after the reaction of the sample is terminated, the control module analyzes the reaction rule of the sample according to the photoelectric signal data received at fixed time intervals, and performs outlier rejection and weighting operation on the photoelectric signal data;
as an example, assuming that the total number of photoelectric signals received by the control module is N from the start of the reaction to the end of the reaction, the signal value obtained at each sampling point is D [i] (0.ltoreq.i.ltoreq.N), (i representing one of N sampling points);
as an illustration, outlier rejection refers to: the total reaction time was set as: 30 minutes, 2 times per minute, each sampling data is X i (i represents one of N sampling points, i is 0.ltoreq.i)<N, in this example n=60), is taken
When |X i -X mean When the I is more than 3 sigma, the abnormal value is removed, and the average value of the data before and after the abnormal value is used for replacing the abnormal value;
as an illustration, the weighted operation refers to: the log of the reaction data over 20 to 30 minutes varies due to the varying degree of the reaction data over 30 minutesThe effect of the value results is more pronounced, so that the data after 10 minutes of reaction is weighted, the acquired data from 10 minutes to 20 minutes is multiplied by 40% weight, the data from 20 minutes to 30 minutes is multiplied by 60% weight, and the weighted data is summed, i.e
Step six, when the numerical result is within the range of the positive threshold value after the operation in the step five, the control module controls the output module to set the positive indicator lamp; when the numerical result is within the negative threshold range, the control module controls the output module 23 to set a negative indicator lamp so as to provide a detection conclusion for a user;
as an illustration, the output module 23 is further configured to output the visualized real-time reaction phenomenon of the sample system and the sample attribute interpretation result;
example 1:
as shown in fig. 4, when the optical filter 12 is not added, in the wavelength band from λ1 to λ2, the spectral line of the sample 1 sampled by the detection module has a certain difference from the spectral line of the sample 2, but in the wavelength band from λ2 to λ3, the spectral line of the sample 1 sampled by the detection module is relatively close to the spectral line of the sample 2, so that the difference between the average value C1 of the sample 1 and the average value C2 of the sample 2 is smaller, and the erroneous judgment of the result is easy to be caused;
example 2:
as shown in fig. 5, the detection value curves of the sample 1 and the sample 2 in the 30-minute constant temperature reaction process are shown; after adding the filtering 12, filtering spectral lines of the lambda 2-lambda 3 wave bands, and only reserving spectral lines of the lambda 1-lambda 2 wave bands, wherein the difference between the average value C1 of the sample 1 sampled by the detection module and the average value C2 of the sample 2 is larger, so that misjudgment is easy;
the equipment provided by the invention is small and exquisite, portable, low in cost, free from special environmental requirements and suitable for household detection; the invention adopts the fully enclosed constant temperature and heat preservation design, so as to prevent liquid from evaporating to the pipe wall and the upper cover during the reaction of the micro sample, and influence the reaction precision; the invention adopts a closed light path design, the indicator lamp can output the interpretation result in the reaction process in real time, replaces human eye interpretation, and reduces errors caused by human eye interpretation;
the foregoing description of the preferred embodiments of the present invention has been presented only to facilitate the understanding of the principles of the invention and its core concepts, and is not intended to limit the scope of the invention in any way, however, any modifications, equivalents, etc. which fall within the spirit and principles of the invention should be construed as being included in the scope of the invention.
Claims (9)
1. A portable loop-mediated isothermal amplification device, comprising: the device comprises a constant temperature heating module, an excitation and detection module, a shell module, a control module and an output module;
the isothermal heating module is used for providing isothermal environment required by sample reaction, and comprises: the device comprises an upper heat cover, a sample constant temperature tank and a base plate;
the excitation and detection module comprises: an excitation module and a detection module; the excitation and detection module is arranged in the substrate, the sample constant temperature tank is provided with an incident light hole and a detection light receiving hole, and the incident light hole and the detection light receiving hole are symmetrically arranged at two ends of the sample constant temperature tank;
the excitation module is used for exciting a luminous group in a sample system and comprises a plurality of excitation subsystems; the excitation subsystem includes: an adjustable excitation light source, an adjustable excitation light source focusing lens; the adjustable excitation light source focusing lens is arranged in front of the adjustable excitation light source, and excitation light irradiates the sample tube through an incident light hole on the sample placing groove;
the detection module is used for receiving the light signal excited by the sample, and is arranged at the detection light receiving hole; the detection module comprises a plurality of detection subsystems; the detection subsystem includes: the optical filter, the detection focusing lens and the photoelectric sensor structure;
the shell module is used for bearing the constant-temperature heating module, the excitation and detection module, the control module and the output module;
the control module is used for adjusting the temperature of the constant-temperature heating module, controlling the excitation and detection module to work, collecting signals of the detection module in real time and judging and reading sample properties;
the output module is arranged at the front end of the shell and used for displaying the interpretation result so as to enable a user to identify the detection result.
2. The portable loop-mediated isothermal amplification device according to claim 1, wherein the upper heat cover is integrated with a first heating material, a first temperature sensor and a first temperature protection switch; an upper heat cover constant temperature protection layer is surrounded on the top and the periphery of the upper heat cover; the sample constant temperature tank is integrated with a second heating material, a second temperature sensor, a second temperature protection switch and the like; the sample constant temperature tank is arranged in the substrate; the bottoms and the peripheries of the sample groove and the substrate are wrapped by a sample constant temperature groove protection layer; during reaction, the reaction system is arranged in a sample tube, and the sample tube is arranged in a constant temperature space between the upper heat cover and the sample constant temperature tank; the first heating material, the first temperature sensor and the first temperature protection switch are electrically connected with the control module.
3. The portable loop-mediated isothermal amplification device according to claim 2, wherein the first and second heat generating materials are: one or a combination of heating films or heating sheets.
4. The portable loop-mediated isothermal amplification device according to claim 1, wherein the number of detection subsystems and excitation subsystems corresponding to the same sample constant temperature tank is equal, and the detection subsystems and the excitation subsystems are bilaterally symmetrical and located at the same liquid level.
5. The portable loop-mediated isothermal amplification device according to claim 2, wherein the housing module comprises: the upper cover, the hinge, the shell, the lock catch and the switch button; the upper cover is connected with the shell through a hinge, and can be opened and closed at 0-90 degrees relative to the shell by taking the hinge as a center, and the lock catch is triggered when the upper cover is closed, so that the upper cover and the shell are locked; the upper heat cover, the first heating material, the first temperature sensor, the first temperature protection switch and the upper heat cover constant temperature protection layer are arranged in the upper cover; the sample constant temperature tank, the second heating material, the second temperature sensor, the second temperature protection switch, the substrate and the sample constant temperature tank protection layer are arranged in the shell.
6. The method for operating a portable loop-mediated isothermal amplification device according to claim 1, comprising:
step one, a control module starts a constant temperature heating module to start heating, and adjusts the temperature according to a feedback value of a temperature sensor so that the reaction temperature of a sample is kept under a constant temperature condition;
step two, the control module controls the excitation module to irradiate excitation light on the sample;
step three, the control module controls the detection module to receive the optical signal generated after the sample is excited, and then the optical signal is subjected to photoelectric conversion and processing to form a photoelectric signal and then transmitted to the control module;
step four, the control module presets the total reaction time, and receives photoelectric signal data according to fixed time intervals in the reaction time; the photoelectric signal data is reaction data;
step five, after the reaction of the sample is terminated, the control module analyzes the reaction rule of the sample according to the photoelectric signal data received at fixed time intervals, and performs outlier rejection and weighting operation on the photoelectric signal data;
the outlier rejection refers to: the total reaction time was set as: 30 minutes, 2 times per minute, each sampling data is X i
Wherein: i represents one of N sampling points, i is more than or equal to 0 and less than or equal to N, N=60, and the sampling points are taken
When |X i -X mean When the I is more than 3 sigma, the abnormal value is removed, and the average value of the data before and after the abnormal value is used for replacing the abnormal value;
the weighting operation is as follows: since the reaction data varies in the degree of 30 minutes, the reaction data in 20 minutes to 30 minutes has more remarkable influence on the numerical result, the data after 10 minutes of reaction is weighted, the collected data of 10 minutes to 20 minutes is multiplied by 40% of weight, the data of 20 minutes to 30 minutes is multiplied by 60% of weight, and the weighted data is summed, that is
Step six, when the numerical result is within the range of the positive threshold after the operation in the step five is completed, the control module controls the output module to set the positive indicator lamp; and when the numerical result is within the negative threshold range, the control module controls the output module to set the negative indicator lamp, so that a detection conclusion is provided for a user.
7. The method of claim 6, wherein the isothermal conditions are: 65 ℃.
8. The method of claim 6, wherein the total number of photoelectric signals received by the control module from the start of the reaction to the end of the reaction is N, and the signal value obtained at each sampling point is D [i] Wherein i is more than or equal to 0 and less than or equal to N, and i represents one of N sampling points.
9. The method according to claim 6, wherein the output module is further configured to output a real-time reaction phenomenon of the visualized sample system and a sample attribute interpretation result.
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| CN113877239B (en) * | 2021-09-30 | 2023-07-25 | 浙江扬清芯片技术有限公司 | Micro-fluidic chip integrating ultrasonic textile extraction and detection |
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