Molecular POCT integrated nucleic acid detection device and method

Abstract

The invention provides a molecular POCT integrated nucleic acid detection device and method, and the device mainly comprises an amplification chamber, a liquid chamber, a detection chamber, a reading chamber, a heating module and a control module, and can further comprise a sample chamber and a sampler. The peristaltic pump is used for quantitatively supplying liquid to the amplification chamber from the liquid chamber, so that the detection error caused by the change of the liquid adding amount is reduced. Meanwhile, a liquid difference channel is designed between the amplification chamber and the detection chamber, and liquid in the amplification chamber is controlled to automatically flow into the detection chamber in a delayed manner by utilizing liquid level difference, so that the automation of the device is improved. According to the device, sample extraction, nucleic acid amplification and detection are integrated, pollution possibly caused by step-by-step operation is reduced, and meanwhile, integration and automation of the device are improved.

Description

Technical Field

[0001] The present invention relates to the field of biochemical analysis, and in particular to a nucleic acid detection device and method for home use. Background Art

[0002] Point-of-care testing (POCT) refers to clinical testing and bedside testing performed next to the patient, which is usually not necessarily performed by a clinical laboratory technician. It is a new method that performs analysis immediately at the sampling site, eliminating the complex handling procedures of the specimen in the laboratory test and quickly obtaining the test results. The main features of POCT are that it does not require a fixed testing site, the reagents and instruments are portable, easy to operate, and can be operated in a timely manner. The most typical POCT products are colloidal gold immunoassay strips.

[0003] Nucleic acid testing can identify specific nucleic acid sequences and detect the presence of pathogen target genes through primer-mediated specific amplification of target genes, thereby providing information and decision-making basis for the diagnosis and treatment of diseases. Nucleic acid testing can shorten the "window period" of diagnosis and can quantitatively detect pathogens. Compared with other traditional methods such as immune detection, it has irreplaceable advantages. At present, the mainstream practice of nucleic acid testing in China still relies on standard PCR laboratories, which are carried out by professional operators holding PCR certificates, and cross-contamination is effectively avoided with the help of PCR partition laboratories.

[0004] In recent years, molecular POCT nucleic acid detection technology has been introduced. The notable features of molecular POCT are that the instrument is small, most of them are desktop, and the nucleic acid extraction and amplification are fully automatic and fully enclosed, so in theory, it can be independent of the PCR laboratory. And because the test results can be given with simple operations, professional operators are no longer required in theory. Molecular POCT nucleic acid detection technology makes home nucleic acid testing possible. In molecular POCT nucleic acid detection technology, nucleic acid extraction and nucleic acid amplification are the two main steps. At present, the home nucleic acid self-test products on the market generally use one-step extraction and constant temperature amplification. Compared with PCR, which requires repeated temperature rise and fall, the reaction process of constant temperature amplification technology is always maintained at a constant temperature, which is easy to operate. At the same time, the temperature required for constant temperature amplification is much lower than the PCR denaturation temperature (95°C). Therefore, the sealing requirements for detection consumables for constant temperature amplification technology are also much lower. The current mainstream constant temperature amplification technologies include loop-mediated constant temperature amplification (LAMP), recombinase polymerase amplification (RPA), rolling circle nucleic acid amplification (RCA), etc.

[0005] However, the current home molecular POCT nucleic acid detection technology still has some shortcomings and needs further improvement. For example, document US20220155294A1 proposes a rapid diagnostic test instrument that can be used at home, including a sample chamber, a first fluid chamber, a second fluid chamber, a test and a readout chamber, wherein the sample chamber and the first fluid chamber, the sample chamber and the second fluid chamber, and the sample chamber and the test and readout chamber are separated by a burstable seal. When in use, the seal is burst by artificial extrusion, so that the buffer of the first fluid chamber enters the sample chamber, and the freeze-dried amplification reagent preset in the sample chamber is activated. The amplification reagent can be a LAMP reagent, an RPA reagent, and a NEAR reagent. After the amplification is completed, the diluent of the second fluid chamber is squeezed into the sample chamber, and finally the liquid in the sample chamber is squeezed to flow into the test and readout chamber, and the test results are displayed on the lateral flow analysis strip. The above scheme has a low degree of automation and requires manual operation. If the liquid cannot flow completely into the sample chamber, the detection accuracy may be affected.

[0006] Document CN114875121A proposes a device and method for detecting nucleic acid amplification products using lateral flow test strips. During the detection, a certain amount of the sample solution to be tested is first added to the dilution closed chamber. Under the action of static pressure, the sample solution to be tested enters the reaction chamber through the channel between the dilution closed chamber and the reaction chamber. It can be a PCR or constant temperature amplification reaction. All reagents required for nucleic acid amplification can be pre-solidified in the reaction chamber; after the amplification reaction is completed, the channel between the reaction chamber and the mixing chamber is opened, and under the action of the static pressure of the solution in the dilution closed chamber, the solution in the reaction chamber enters the mixing chamber; the sample solution that has not been amplified in the dilution closed chamber also passes through the reaction chamber and further enters the mixing chamber until the water pressure in the dilution closed chamber, the reaction chamber and the mixing chamber is balanced. The test results are displayed on the nucleic acid lateral flow test strip in the mixing chamber. The above scheme also has the problem of low degree of automation and difficulty in accurately controlling the amount of liquid.

[0007] Therefore, there is still a need to develop a new molecular POCT nucleic acid detection device and corresponding method to better realize home nucleic acid detection. Summary of the invention

[0008] The present invention mainly provides a molecular POCT nucleic acid detection device and method based on constant temperature amplification technology, which integrates nucleic acid extraction and amplification, and can quickly and conveniently perform sample lysis, nucleic acid amplification and detection.

[0009] The present invention provides a first molecular POCT integrated nucleic acid detection device, comprising:

[0010] an amplification chamber, wherein the amplification chamber has an opening for receiving a sample, and a nucleic acid amplification reagent is pre-cured in the amplification chamber;

[0011] The liquid chamber is connected to the amplification chamber through a first pipeline, a first peristaltic pump is provided on the first pipeline, and a diluent is stored in the liquid chamber;

[0012] The detection chamber is connected to the amplification chamber through a second pipeline, and a second peristaltic pump is provided on the second pipeline; or the detection chamber is connected to the amplification chamber through a liquid level difference channel, and when the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by the liquid level difference, and an amplification termination reagent is pre-solidified in the detection chamber;

[0013] A readout chamber in a vertical direction, the bottom of which is directly connected to the detection chamber, and a lateral flow test strip is arranged inside the readout chamber;

[0014] A heating module, located at the periphery of the amplification chamber, having an amplification temperature and a detection temperature;

[0015] A control module controls the heating module, the first peristaltic pump or the first peristaltic pump and the second peristaltic pump.

[0016] Preferably, the detection chamber is connected to the amplification chamber through a liquid level difference channel.

[0017] The detection method of the first device is as follows: adding a sample into the amplification chamber, the control module controls the first peristaltic pump to rotate, pumping at least a portion of the diluent in the liquid chamber into the amplification chamber, and forming an amplification liquid with the pre-solidified nucleic acid amplification reagent in the amplification chamber, and at the same time controlling the heating module to be at the amplification temperature; after the heating time reaches the set time, the control module controls the heating module to stop heating or switch to the detection temperature, and at the same time controls the second peristaltic pump to rotate, so that all the amplification liquid in the amplification chamber is pumped into the detection chamber, or controls the first peristaltic pump to rotate, pumping the remaining portion of the diluent in the liquid chamber into the amplification chamber, so that the liquid level in the amplification chamber rises and the amplification liquid flows into the detection chamber through the liquid level difference channel; the amplification liquid flowing into the detection chamber contacts with the pre-solidified amplification termination reagent in the detection chamber to form a detection liquid; the detection liquid contacts the lateral flow test strip to perform chromatographic reaction detection.

[0018] The present invention provides a second molecular POCT integrated nucleic acid detection device, comprising:

[0019] a sample chamber having an opening for receiving a sample;

[0020] an amplification chamber, located below the sample chamber and separated from the sample chamber by a puncturable sealing membrane;

[0021] The liquid chamber is connected to the amplification chamber through a first pipeline, and a first peristaltic pump is provided on the first pipeline;

[0022] The detection chamber is connected to the amplification chamber through a liquid level difference channel. When the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by the liquid level difference.

[0023] A readout chamber in a vertical direction, the bottom of which is directly connected to the detection chamber, and a lateral flow test strip is arranged inside the readout chamber;

[0024] A heating module, located at the periphery of the amplification chamber, having an amplification temperature and a detection temperature;

[0025] A control module controls the heating module and the first peristaltic pump.

[0026] Preferably, a diluent is stored in the sample chamber, a nucleic acid amplification reagent is pre-solidified in the amplification chamber, and an amplification termination solution is stored in the liquid chamber.

[0027] Preferably, a diluent is stored in the sample chamber, a diluent is stored in the liquid chamber, a nucleic acid amplification reagent is pre-solidified in the amplification chamber, and an amplification termination reagent is pre-solidified in the detection chamber.

[0028] The detection method of the second device is as follows: add the sample into the sample chamber, puncture the sealing diaphragm between the sample chamber and the amplification chamber, so that the sample and the diluent in the sample chamber all enter the amplification chamber, and form an amplification liquid with the pre-solidified nucleic acid amplification reagent in the amplification chamber; the control module controls the heating module to be at the amplification temperature, and after the heating time reaches the set time, the control module controls the heating module to stop heating or change to the detection temperature, and at the same time controls the first peristaltic pump to rotate, pumping the liquid in the liquid chamber into the amplification chamber; the liquid in the amplification chamber flows into the detection chamber by itself through the liquid level difference channel; the liquid contacts the lateral chromatography test strip to perform chromatography reaction detection.

[0029] The present invention provides a third molecular POCT integrated nucleic acid detection device, comprising:

[0030] The sampler has a hollow pipe in which a diluent is sealed. The head of the sampler is a porous structure, and the tail end of the sampler is a sealing film. After the sealing film is punctured, the diluent flows out from the head.

[0031] an amplification chamber having an opening for receiving a sample;

[0032] The liquid chamber is connected to the amplification chamber through a first pipeline, and a first peristaltic pump is provided on the first pipeline;

[0033] The detection chamber is connected to the amplification chamber through a liquid level difference channel. When the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by the liquid level difference.

[0034] A readout chamber in a vertical direction, the bottom of which is directly connected to the detection chamber, and a lateral flow test strip is arranged inside the readout chamber;

[0035] A heating module, located at the periphery of the amplification chamber, having an amplification temperature and a detection temperature;

[0036] A control module controls the heating module and the first peristaltic pump.

[0037] Preferably, a nucleic acid amplification reagent is pre-solidified in the amplification chamber, and an amplification termination solution is stored in the liquid chamber.

[0038] Preferably, a nucleic acid amplification reagent is pre-solidified in the amplification chamber, a diluent is stored in the liquid chamber, and an amplification termination reagent is pre-solidified in the detection chamber.

[0039] The detection method of the third device is as follows: place the head of the sampler with the sample in the amplification chamber, puncture the sealing film at the tail end of the sampler, and allow the diluent inside the sampler to flow out from the head of the sampler to form an amplification liquid with the pre-solidified nucleic acid amplification reagent in the amplification chamber; the control module controls the heating module to be at the amplification temperature, and after the heating time reaches the set time, the control module controls the heating module to stop heating or change to the detection temperature, and at the same time controls the first peristaltic pump to rotate to pump the liquid in the liquid chamber into the amplification chamber; the liquid in the amplification chamber flows into the detection chamber by itself through the liquid level difference channel; the liquid contacts the lateral chromatography test strip to perform chromatography reaction detection.

[0040] The present invention adopts RPA isothermal amplification technology. Compared with other isothermal amplification technologies, RPA has a low amplification reaction temperature and has the advantages of high sensitivity, selectivity, portability, rapidity, and multiple amplification, and is suitable for molecular POCT technology. The present invention adds labeled probes and nucleases to the RPA system to perform real-time detection; combined with lateral flow technology, visual detection can be achieved, which is suitable for the POCT field.

[0041] The advantages of the present invention are as follows:

[0042] 1) The quantitative addition of diluent or stop solution is achieved through a micro peristaltic pump, reducing the detection error caused by changes in the amount of liquid added;

[0043] 2) Design a liquid differential channel between the amplification chamber and the detection chamber, and use the liquid level difference to control the automatic and delayed flow of the liquid in the amplification chamber into the detection chamber, thereby improving the automation of the device;

[0044] 3) Integrating sample extraction, nucleic acid amplification and detection into one device reduces the contamination that may be caused by step-by-step operations, while improving the integration and automation of the device. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the first embodiment of the present invention.

[0046] Figures 2A-2C It is a schematic diagram of a second embodiment of the present invention.

[0047] Figures 3A-3D It is a schematic diagram of a third embodiment of the present invention.

[0048] Figures 4A-4D It is a schematic diagram of a fourth embodiment of the present invention.

[0049] in:

[0050] 100, 100', 200, 300: device housing;

[0051] 101, 101', 201, 301: liquid chamber;

[0052] 102, 102', 202, 302a, 302b: sample diluent;

[0053] 103, 103': liquid level of sample dilution solution in initial state;

[0054] 104, 104', 204, 304: amplification chamber;

[0055] 105a, 105b, 105', 205, 305: micro peristaltic pump;

[0056] 106, 106', 206, 306: testing room;

[0057] 107, 107', 207, 307: Lateral flow test strips;

[0058] 108, 108', 208, 308: Reading room;

[0059] 109, 109', 209, 309: Amplification lyophilized reagents;

[0060] 209A, 309A: amplification solution obtained by dissolving amplification freeze-dried reagent;

[0061] 209B, 309B: detection solution obtained by mixing the stop dilution solution;

[0062] 110, 110', 310: stop freeze-drying reagent;

[0063] 213: stop dilution;

[0064] 111, 111', 211, 311: heating modules;

[0065] 112', 212, 312: liquid level difference channel;

[0066] 214, 314: sample room;

[0067] 214A, 314A: Sealing cover;

[0068] 314B: needle;

[0069] 314C: connection portion between the amplification chamber and the sample chamber;

[0070] 214B: diaphragm;

[0071] 215, 315: sampler;

[0072] 215A, 315A: sampling head;

[0073] 316: Reading window. DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described below according to specific embodiments. The protection scope of the present invention is not limited to the following embodiments, which are listed only for illustrative purposes and do not limit the present invention in any way.

[0075] The present invention is further described in detail below by way of examples.

[0076] The micro peristaltic pump used in the embodiment of the present invention is PYRP310-XA of Pincheng Motor, which can also be replaced by Kamoer KPP-DC-B08W. The side used for the liquid channel in the liquid chamber, amplification chamber, detection chamber and readout chamber is made of plastic, and the other sides are mainly made of flexible materials, such as metal foil bags, polymer bags and other materials. The overall size of the device is about half the size of a mobile phone case. The control module uses an STM32 chip.

[0077] Example 1

[0078] Figure 1 A molecular POCT integrated nucleic acid detection device of the present invention is shown, including a liquid chamber 101, an amplification chamber 104, a detection chamber 106, a readout chamber 108, micro peristaltic pumps 105a and 105b, a heating module 111 and a control module (not shown). The liquid chamber 101 is connected to the amplification chamber 104 through a pipeline, and a micro peristaltic pump 105a is provided on the pipeline. The detection chamber 106 is connected to the amplification chamber 104 through a pipeline, and a micro peristaltic pump 105b is provided on the pipeline. The readout chamber 108 is in a vertical direction and is directly connected to the detection chamber 106 at the bottom. The heating module 111 is located on the periphery of the amplification chamber 104 and has an amplification temperature and a detection temperature. The control module can control the heating module 111 to be at an amplification temperature or a detection temperature, and at the same time control the operation of the micro peristaltic pumps 105a and 105b.

[0079] The heating module maintains a constant temperature of 42.0±1.0℃. The heating module can be separated from the detection module device and is an independent module. When heating is required, the detection device can be inserted through a socket for heating. The heating range of the constant temperature heating module covers the amplification chamber and the detection chamber. When the device is in the amplification period, the temperature used by the module is the amplification temperature. When the device completes the amplification and enters the detection period, the heating module is adjusted to the detection temperature through electrical control, which can complete the whole process of temperature control, making the temperature of the entire detection process controllable, thereby reducing the result variation caused by temperature during the POCT detection process.

[0080] When not in use, the liquid chamber 101 stores a sample diluent 102. The liquid chamber 101 has only one outlet connected to the pipeline of the micro peristaltic pump 105a, and the other areas are sealed. A small section of air is left in the pipeline to ensure that the atmospheric pressure and liquid tension can lock the liquid in the room and will not leak out due to shaking when it is not in operation. The material of the liquid chamber is a flexible material, which can be a metal foil bag, a polymer bag, etc., and the volume of liquid that can be accommodated is 500 to 1000 microliters. The amplification chamber 104 is pre-cured with an amplification freeze-dried reagent 109. The amplification freeze-dried reagent 109 is mainly a freeze-dried reagent obtained by freeze-drying technology for the enzymes, primers, probes, and buffer ions required for amplification. After dissolution, it can restore activity and perform an amplification reaction with the target nucleic acid sequence in the sample at a certain constant temperature. The detection chamber 106 is pre-cured with a termination freeze-dried reagent 110. The termination freeze-dried reagent 110 is mainly salt ions that terminate the amplification reaction, which are also obtained by freeze-drying technology. A lateral flow test strip 107 is fixed inside the reading chamber 108. The test strip can be any one of a colloidal gold lateral flow test strip, a latex lateral flow test strip, a chemiluminescent lateral flow test strip, and a time-resolved fluorescence lateral flow test strip. The reading chamber is in a sealed and dry state to ensure the effectiveness of the test strip in the chamber.

[0081] In this embodiment, the micro peristaltic pump 105a is used to connect the liquid chamber 102 and the amplification chamber 104, and the micro peristaltic pump 105b is used to connect the amplification chamber 104 and the detection chamber 106. When in use, after the sample diluent 102 enters the amplification chamber 104 through the micro peristaltic pump 105a, the amplification freeze-dried reagent 109 is dissolved, and the heating module 111 is started to keep the amplification chamber 104 at the amplification temperature. The heating module can set the heating temperature and working time. When the time is up, the heating module switches to the detection temperature, and the micro peristaltic pump 105b works at the same time to pump the liquid after amplification into the detection chamber 106, and dissolve the termination freeze-dried reagent 110 at the same time, and the amplification reaction is terminated. When the liquid level in the detection chamber reaches a certain height, the liquid level contacts the lateral chromatography test strip 107 in the reading chamber 108, and the liquid climbs up along the test strip, and finally reaches the detection area of ​​the test strip and displays the result.

[0082] Example 2

[0083] Figure 2A Another molecular POCT integrated nucleic acid detection device of the present invention is shown, including a liquid chamber 101', an amplification chamber 104', a detection chamber 106', a readout chamber 108', a micro peristaltic pump 105', a heating module 111' and a control module (not shown). The liquid chamber 101' is connected to the amplification chamber 104' through a pipeline, and a micro peristaltic pump 105' is provided on the pipeline. The detection chamber 106' is connected to the amplification chamber 104' through a liquid level difference channel 112'. When the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by itself through the liquid level difference. The readout chamber 108' is in a vertical direction and is directly connected to the detection chamber 106' at the bottom. The heating module 111' is located on the periphery of the amplification chamber 104' and has an amplification temperature and a detection temperature. The control module can control the heating module 111' to be at an amplification temperature or a detection temperature, and at the same time control the operation of the micro peristaltic pump 105'.

[0084] When not in use, the liquid chamber 101' stores a sample diluent 102'. The amplification chamber 104' is pre-cured with an amplification lyophilized reagent 109', and the detection chamber 106' is pre-cured with a termination lyophilized reagent 110'. The readout chamber 108' has a lateral flow test strip 107' fixed therein.

[0085] refer to Figure 2B When in use, after the first part of the sample diluent 102' enters the amplification chamber 104' through the micro peristaltic pump 105', the amplification freeze-dried reagent 109' is dissolved. At this time, the liquid level in the amplification chamber 104' is 103A'. Due to the design of the liquid level difference channel 112' on the horizontal plane, it does not touch the detection chamber. Then the heating module 111' is started to keep the amplification chamber 104' at the amplification temperature. The heating module can set the working time. When the time is up, the heating module switches to the detection temperature. Reference Figure 2C , the micro peristaltic pump 105' continues to work, pumping the second part of the sample diluent 102' into the amplification chamber 104', raising the liquid level 103A' in the amplification chamber 104' to a level where the liquid can enter the detection chamber 106', and dissolving the freeze-dried reagent 110' to form a detection liquid. When the liquid level in the detection chamber reaches a certain height, the liquid surface contacts the lateral flow test strip 107' in the reading chamber 108', and the liquid climbs up along the test strip, finally reaching the detection area of ​​the test strip and displaying the result.

[0086] Regarding the amount of the first part of the sample diluent and the second part of the sample diluent, the first part of the sample diluent only needs to be pumped into the amplification chamber 104' in a volume of about 50 to 200 microliters. Here, the amplification diluent should not be too much. The more diluent, the more lyophilized reagent 109' will be needed, and the cost will be higher; the second part of the sample diluent is about 5 times the first part of the sample diluent, that is, if the first part of the sample diluent is 50 microliters, the second part of the sample diluent should be pumped in about 250 microliters. The liquid in the liquid chamber 101' can be excessive, and the volume of liquid flowing in can be controlled by a micro peristaltic pump.

[0087] Example 3

[0088] Figure 3A Another molecular POCT integrated nucleic acid detection device of the present invention is shown, and compared with the first two embodiments, a sample chamber is added. The device includes a sample chamber 214, a liquid chamber 201, an amplification chamber 204, a detection chamber 206, a readout chamber 208, a micro peristaltic pump 205, a heating module 211 and a control module (not shown). The sample chamber 214 has a sealing cover 214A for receiving a sample. The amplification chamber 204 is located below the sample chamber 214 and is separated from the sample chamber 214 by a puncturable sealing diaphragm 214B. The liquid chamber 201 is connected to the amplification chamber 204 through a pipeline, and a micro peristaltic pump 205 is provided on the pipeline. The detection chamber 206 is connected to the amplification chamber 204 through a liquid level difference channel 212. When the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by itself through the liquid level difference. The readout chamber 208 is in a vertical direction and is directly connected to the detection chamber 206 below. The heating module 211 is located at the periphery of the amplification chamber 204 and has an amplification temperature and a detection temperature. The control module can control the heating module 211 to be at an amplification temperature or a detection temperature, and control the operation of the micro peristaltic pump 205 at the same time.

[0089] When not in use, the sample chamber 214 stores a sample diluent 202, and the liquid chamber 201 stores a stop diluent 213. A freeze-dried amplification reagent 209 is pre-solidified in the amplification chamber 204. A lateral flow test strip 207 is fixed in the reading chamber 208.

[0090] refer to Figure 3B When in use, after the sampler 215 collects the sample, the sample is attached to the sampling head 215A, the sealing cover 214A of the sample chamber 214 is opened, and the sampling head 215A is completely immersed in the sample diluent 202, and the sample diluent can release the analyte in the sample.

[0091] refer to Figure 3C, force is applied to make the sampling head 215A break the diaphragm 214B, so that the sample diluent containing the extracted sample flows into the amplification chamber 204 below, and dissolves the amplification freeze-dried reagent 209 to become the amplification liquid 209A. At this time, the liquid level of the amplification liquid is blocked by the liquid level difference channel 212 and is not enough to enter the detection chamber. The heating module 211 is started to provide a suitable amplification temperature for the liquid in the amplification chamber, and a certain heating time is set. When the time is up, it is switched to the detection temperature.

[0092] refer to Figure 3D After the amplification is completed, the micro peristaltic pump 205 is operated to pump the termination diluent 213 in the liquid chamber 201 into the amplification chamber to mix with the amplification liquid 209A and dilute it to form the detection liquid 209B. The liquid level of the detection liquid is higher than the liquid level difference channel 212, so that the liquid enters the detection chamber 206. As the liquid level rises, it reaches the sample pad at the bottom of the lateral flow test strip 207 and is absorbed and climbs up, and the result is read out through the test strip.

[0093] Example 4

[0094] Figure 4A Another molecular POCT integrated nucleic acid detection device of the present invention is shown, which includes a sampler 315, a sample chamber 314, a liquid chamber 301, an amplification chamber 304, a detection chamber 306, a readout chamber 308, a micro peristaltic pump 305, a heating module 311 and a control module (not shown).

[0095] The sample chamber 314 has a sealing cover 314A for receiving the sample, and the sealing cover 314A is provided with a lancet 314B. The amplification chamber 304 is located below the sample chamber 314 and is directly connected to the sample chamber. The connection 314C between the amplification chamber and the sample chamber is relatively narrow, and the width is just enough to insert the sampler, which plays the role of fixing the sampler. The liquid chamber 301 is connected to the amplification chamber 304 through a pipeline, and a micro peristaltic pump 305 is provided on the pipeline. The detection chamber 306 is connected to the amplification chamber 304 through a liquid level difference channel 312. When the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by itself through the liquid level difference. The reading chamber 308 is in a vertical direction and is directly connected to the detection chamber 306 at the bottom. The heating module 311 is located at the periphery of the amplification chamber 304 and has an amplification temperature and a detection temperature. The control module can control the heating module 311 to be at the amplification temperature or the detection temperature, and at the same time control the operation of the micro peristaltic pump 305.

[0096] When not in use, the interior of the sampler 315 is a hollow pipe sealed with a sample diluent 302a, one end of which is a sampling head 315A, and the tail end is a sealing film (not shown), so that the sample diluent inside the sampler is firmly adsorbed in the pipe due to the air pressure. The material used by the sampling head 315A is a material with pores, which can be a flocking head (polyamide), a sponge head (polyurethane), a synthetic fiber swab (acetate fiber, polyester fiber, etc.), and non-woven fabrics, polyester fabrics, etc. can also be used according to different sampling requirements. When the sealing film is punctured by the needle 314B on the sealing cover 314A through the cover pressure, air enters the cavity, causing the original air pressure to change, and the sample diluent 302a in the tube is discharged from the pores of the sampling head below the tube, and at the same time, the sample collected outside the sampling head is flushed out or dissolved.

[0097] The liquid chamber 301 stores a sample diluent 302b. The amplification chamber 304 pre-solidifies an amplification lyophilized reagent 309, and the detection chamber 306 pre-solidifies a termination lyophilized reagent 310. The readout chamber 308 is fixed with a lateral flow test strip 307. Of course, the sample diluent in the liquid chamber can also be replaced with a termination diluent, in which case, the termination lyophilized reagent does not need to be solidified in the detection chamber.

[0098] There is a reading window 316 in the middle of the reading chamber 308, and its position just corresponds to the position of the detection area (ie, the CT line) of the lateral flow test strip, so that the result can be directly viewed.

[0099] Further references Figure 4A When in use, after the sampler 315 takes a sample, the sealing cover 314A is opened and the sampler with the sample is inserted into the sampler and fixed. At this time, the sealing film at the tail end of the sampler is just at the opening of the sampler.

[0100] refer to Figure 4B When the sealing cover 314A is re-covered on the opening of the sampler, the needle 314B in the sealing cover pierces the sealing film at the tail end of the sampler, and air flows into the sampler lumen, causing the sample diluent therein to flow out from the sampling head, flushing out the collected sample and releasing the nucleic acid. At the same time, the sample diluent dissolves the amplification freeze-dried reagent at the bottom of the amplification chamber 304 to form an amplification solution 309A. At this moment, the heating module 311 is started to provide temperature conditions for amplification, and a certain heating time is set. When the time is up, it switches to the detection temperature.

[0101] refer to Figure 4C After the amplification is completed, the micro peristaltic pump 305 is started to pump the liquid in the liquid chamber 301 into the amplification chamber 304, so that the liquid in the amplification chamber increases, and the liquid level rises to cover the liquid level difference channel and reaches the detection chamber 306 to form the detection liquid 309B.

[0102] refer to Figure 4D When the liquid surface of the detection liquid contacts the sample pad of the chromatographic test strip 307, the liquid surface moves upward along the test strip, and when it passes through the reading window 316 corresponding to the result display area of ​​the test strip, the test strip will display the detection result.

[0103] The molecular POCT integrated nucleic acid detection device provided in the above embodiments has a simple structural design, realizes quantitative addition of liquid through a micro peristaltic pump, and improves the automation of the device through a liquid differential channel, which is conducive to application in home nucleic acid detection.

Claims

1. A molecular POCT integrated nucleic acid detection device, characterized in that: include: an amplification chamber having an opening for receiving a sample; a liquid chamber connected to the amplification chamber via a first pipeline, wherein a first peristaltic pump is provided on the first pipeline; The detection chamber is connected to the amplification chamber through a second pipeline, and a second peristaltic pump is provided on the second pipeline; or the detection chamber is connected to the amplification chamber through a liquid level difference channel, and when the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by the liquid level difference; A readout chamber in a vertical direction, wherein the bottom of the readout chamber is directly connected to the detection chamber, and a lateral flow test strip is arranged inside the readout chamber; A heating module, located at the periphery of the amplification chamber, having an amplification temperature and a detection temperature; A control module controls the heating module, the first peristaltic pump, or the first peristaltic pump and the second peristaltic pump.

2. The molecular POCT integrated nucleic acid detection device according to claim 1, characterized in that: The detection chamber is communicated with the amplification chamber through a liquid level difference channel.

3. The molecular POCT integrated nucleic acid detection device according to claim 1, characterized in that: The liquid chamber stores a diluent, the amplification chamber contains a nucleic acid amplification reagent pre-solidified, and the detection chamber contains an amplification termination reagent pre-solidified.

4. A molecular POCT integrated nucleic acid detection device, characterized in that: include: a sample chamber having an opening for receiving a sample; an amplification chamber, located below the sample chamber and separated from the sample chamber by a puncturable sealing membrane; a liquid chamber connected to the amplification chamber via a first pipeline, wherein a first peristaltic pump is provided on the first pipeline; The detection chamber is connected with the amplification chamber through a liquid level difference channel, and when the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by the liquid level difference; A readout chamber in a vertical direction, wherein the bottom of the readout chamber is directly connected to the detection chamber, and a lateral flow test strip is arranged inside the readout chamber; A heating module, located at the periphery of the amplification chamber, having an amplification temperature and a detection temperature; A control module controls the heating module and the first peristaltic pump.

5. The molecular POCT integrated nucleic acid detection device according to claim 4, characterized in that: The sample chamber stores a diluent, the amplification chamber contains a nucleic acid amplification reagent that is pre-solidified, and the liquid chamber stores an amplification termination solution.

6. The molecular POCT integrated nucleic acid detection device according to claim 4, characterized in that: The sample chamber stores a diluent, the amplification chamber pre-solidifies a nucleic acid amplification reagent, the liquid chamber stores a diluent, and the detection chamber pre-solidifies an amplification termination reagent.

7. A molecular POCT integrated nucleic acid detection device, characterized in that: include: The sampler has a hollow pipe, in which a diluent is sealed, the head of the sampler is a porous structure, and the tail end of the sampler is a sealing film. After the sealing film is punctured, the diluent flows out from the head; an amplification chamber having an opening for receiving a sample; a liquid chamber connected to the amplification chamber via a first pipeline, wherein a first peristaltic pump is provided on the first pipeline; The detection chamber is connected with the amplification chamber through a liquid level difference channel, and when the liquid in the amplification chamber reaches a certain height, it flows into the detection chamber by the liquid level difference; A readout chamber in a vertical direction, wherein the bottom of the readout chamber is directly connected to the detection chamber, and a lateral flow test strip is arranged inside the readout chamber; A heating module, located at the periphery of the amplification chamber, having an amplification temperature and a detection temperature; A control module controls the heating module and the first peristaltic pump.

8. The molecular POCT integrated nucleic acid detection device according to claim 7, characterized in that: Nucleic acid amplification reagents are pre-solidified in the amplification chamber, and amplification termination liquid is stored in the liquid chamber.

9. The molecular POCT integrated nucleic acid detection device according to claim 7, characterized in that: A nucleic acid amplification reagent is pre-solidified in the amplification chamber, a diluent is stored in the liquid chamber, and an amplification termination reagent is pre-solidified in the detection chamber.

10. A detection method applied to the device according to any one of claims 1 to 3, characterized in that: A sample is added into the amplification chamber, and the control module controls the first peristaltic pump to rotate, pumping at least a portion of the diluent in the liquid chamber into the amplification chamber to form an amplification solution with the pre-solidified nucleic acid amplification reagent in the amplification chamber, and at the same time controlling the heating module to be at the amplification temperature; After the heating time reaches the set time, the control module controls the heating module to stop heating or switch to the detection temperature, and at the same time controls the second peristaltic pump to rotate so that all the amplification liquid in the amplification chamber is pumped into the detection chamber, or controls the first peristaltic pump to rotate to pump the remaining dilution liquid in the liquid chamber into the amplification chamber, so that the liquid level in the amplification chamber rises and the amplification liquid flows into the detection chamber through the liquid level difference channel; The amplification solution flowing into the detection chamber contacts with the amplification termination reagent pre-solidified in the detection chamber to form a detection solution; The detection liquid contacts the lateral flow test strip to perform chromatographic reaction detection.

11. A detection method applied to the device according to any one of claims 4 to 6, characterized in that: Add the sample into the sample chamber, puncture the sealing membrane between the sample chamber and the amplification chamber, so that the sample and the diluent in the sample chamber all enter the amplification chamber, and form an amplification solution with the pre-solidified nucleic acid amplification reagent in the amplification chamber; The control module controls the heating module to be at the amplification temperature. After the heating time reaches the set time, the control module controls the heating module to stop heating or change to the detection temperature, and at the same time controls the first peristaltic pump to rotate to pump the liquid in the liquid chamber into the amplification chamber; The liquid in the amplification chamber flows into the detection chamber by itself through the liquid level difference channel; The liquid contacts the lateral flow test strip and performs chromatographic reaction detection.

12. A detection method applied to the device according to any one of claims 7 to 9, characterized in that: Place the head of the sampler with the sample in the amplification chamber, puncture the sealing film at the tail end of the sampler, and allow the diluent inside the sampler to flow out from the head of the sampler to form an amplification solution with the pre-solidified nucleic acid amplification reagent in the amplification chamber; The control module controls the heating module to be at the amplification temperature. After the heating time reaches the set time, the control module controls the heating module to stop heating or change to the detection temperature, and at the same time controls the first peristaltic pump to rotate to pump the liquid in the liquid chamber into the amplification chamber; The liquid in the amplification chamber flows into the detection chamber by itself through the liquid level difference channel; The liquid contacts the lateral flow test strip and performs chromatographic reaction detection.

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