Fully-integrated multiplex nucleic acid quantitative detection device

Abstract

The utility model provides a fully integrated multiplex nucleic acid quantitative detection device which comprises chambers and channels, the chambers comprise at least one sample cracking chamber, at least one nucleic acid cleaning chamber, at least two reaction chambers and at least two quantitative chambers, the sample cracking chamber, the nucleic acid cleaning chamber, the reaction chamber and the quantitative chamber are communicated through the channels, at least one partition plate is arranged at the bottom of the sample cracking chamber or the bottom of the nucleic acid cleaning chamber, and a plurality of micro reaction units are arranged in the quantitative chamber. The device is turned over by 90 degrees, and the reagent in the reaction chamber can be sucked by an external pump or enter the micro reaction units in the quantitative chamber based on a self-driving mode. A channel switch position is arranged on the channel, a pressure part is arranged at the channel switch position, and reaction reagents in the cavities can be prevented from being mixed by applying pressure to the channel through the pressure part. The device has the beneficial effects that the device is simple in structural design, convenient to operate and easy to carry, liquid does not need to be moved basically, and multiple nucleic acid quantitative analysis can be carried out.

Description

Technical Field

[0001] The utility model relates to the technical field of detection and analysis, and in particular to a fully integrated multiple nucleic acid quantitative detection device. Background Art

[0002] Nucleic acid detection technology has been widely used in disease diagnosis, food safety testing, environmental monitoring and other fields due to its high sensitivity and high specificity. The nucleic acid detection process usually includes steps such as nucleic acid extraction, nucleic acid amplification, and product detection, which require repeated pipetting operations, and cross-contamination of reagents may occur during the operation, affecting the experimental results. In order to make the operation more convenient, researchers have developed some integrated nucleic acid analysis devices. Chinese patent CN110075935 B relates to a multi-index detection microfluidic cartridge and an application method, which uses a syringe combined with a valve to achieve liquid movement between different chambers to complete integrated nucleic acid analysis, but requires repeated pipetting and cannot perform multiple quantitative analyses. Chinese patent CN 105316224 B relates to a fully automatic nucleic acid extraction and PCR amplification microfluidic chip, which uses rotary centrifugation to push the liquid to flow in the microchannel, and has high requirements for the speed control of the supporting instrument. Chinese patent CN 114292734 B relates to a fully integrated droplet digital PCR chip, preparation method, and application. This chip utilizes droplets combined with PCR for integrated quantitative detection, requiring the use of temperature control instruments. The uniformity of droplet size generation is difficult to control, and droplets are also unstable under high-temperature conditions, making multiplex analysis impossible. Chinese patent CN 116103139 B relates to a fully enclosed, fully integrated nucleic acid detection microfluidic chip. This requires dedicated instrumentation, resulting in a complex structural design and high cost, and inability to perform multiplex quantitative detection. Chinese patent CN 107129930 B relates to a fully integrated nucleic acid detection microfluidic chip and its use method. This chip utilizes numerous pump and valve structures, resulting in complex chip fabrication and high cost. Chinese patent CN 114931986 B relates to a nucleic acid detection microfluidic device and nucleic acid detection method. The chip's structural design is also complex, and it can only perform qualitative nucleic acid analysis. Currently, some integrated nucleic acid detection systems are commercially available, but most operate by driving liquids, resulting in relatively complex structures and expensive supporting instruments.

[0003] The CRISPR / Cas system is an adaptive immune system found in bacteria and archaea, primarily composed of Cas proteins and corresponding crRNAs. With the discovery of the bypass cleavage effect of some Cas proteins, some researchers have established novel nucleic acid detection platforms based on the CRISPR / Cas system. When a target nucleic acid is present, guided by crRNA, the Cas protein and crRNA complex precisely recognize and bind to the target nucleic acid, thereby stimulating the bypass cleavage activity of the Cas protein, cleaving the single-stranded signal nucleic acid to produce a detection signal. Due to its excellent sensitivity and specificity, the CRISPR / Cas system has gradually been used for nucleic acid detection. Chinese Patent CN111235232 B discloses a visual rapid nucleic acid detection method and application based on the CRISPR / Cas12a system. However, it can only perform qualitative nucleic acid detection and requires multiple liquid pipetting during the operation, which is a complex process. Chinese Patent CN108823291 B discloses a method for quantitatively detecting specific nucleic acid fragments based on CRISPR technology. However, this method cannot complete sample nucleic acid pretreatment and requires a droplet generation device, which increases the complexity of the device.

[0004] Nucleic acid quantitative testing is extremely important, especially in the fields of disease diagnosis and food safety testing. For example, the diagnosis of many diseases requires knowing the nucleic acid load to determine the severity of the disease and provide appropriate treatment plans. Quantitative nucleic acid testing for pathogenic microorganisms in food is a key indicator of food hygiene quality and can accurately assess the degree of food contamination.

[0005] Therefore, it is of great significance to develop an integrated nucleic acid detection device with low production cost, simple structural design, less liquid drive, and the ability to perform multiple quantitative analyses. This device can be combined with the CRISPR / Cas system to carry out multiple nucleic acid quantitative analyses. Utility Model Content

[0006] The utility model provides a fully integrated multiple nucleic acid quantitative detection device, comprising a chamber and a channel, wherein the chamber comprises at least one sample lysis chamber, at least one nucleic acid cleaning chamber, at least two reaction chambers, and at least two quantitative chambers, the chambers being connected through the channel, at least one partition being provided at the bottom of the sample lysis chamber or the bottom of the nucleic acid cleaning chamber, and a plurality of tiny reaction units being contained in the quantitative chamber; a channel switch position is provided on the channel, and a pressure component is provided at the channel switch position, and applying pressure to the channel by the pressure component can prevent the reaction reagents between the chambers from mixing with each other.

[0007] As a further improvement of the present invention, the channel is arranged in the middle area of ​​the chamber or the top area of ​​the chamber.

[0008] As a further improvement of the present invention, the chamber and the channel are made of a hard material and a flexible material, and the hard material and the flexible material are assembled together.

[0009] As a further improvement of the present invention, the pressure component is a clamp, or the pressure component is a physical valve or a surface tension valve.

[0010] As a further improvement of the present invention, the reaction chamber includes a first reaction chamber and a second reaction chamber, and reagent addition ports are provided on the tops of the sample lysis chamber, the nucleic acid cleaning chamber, the first reaction chamber and the second reaction chamber, and tube caps are installed at the reagent addition ports.

[0011] As a further improvement of the present invention, the tube cap is a physical sealing cover, or the tube cap is a diaphragm that can allow air to pass through but can prevent nucleic acid molecules from passing through.

[0012] As a further improvement of the present invention, the fully integrated multiple nucleic acid quantitative detection device also includes an external pump, the quantitative chamber includes a first quantitative chamber and a second quantitative chamber, the first quantitative chamber and the second quantitative chamber are jointly provided with an interface, and the first quantitative chamber and the second quantitative chamber are connected to the external pump through the interface.

[0013] As a further improvement of the present invention, the external pump includes a syringe pump, an air pump, and a plunger pump.

[0014] As a further improvement of the present invention, a heating device is provided below the first quantitative chamber and the second quantitative chamber.

[0015] As a further improvement of the present invention, the hard material is a polymethyl methacrylate acrylic plate, and the flexible material is a high-temperature resistant acrylic tape.

[0016] The beneficial effects of the utility model are as follows: the utility model has a simple structural design, is easy to operate, is easy to carry, requires less liquid drive, and can perform multiple nucleic acid quantitative analyses. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the structure of a fully integrated multiple nucleic acid quantitative detection device of the present invention (the channel is arranged in the top area of ​​the chamber);

[0018] Figure 2 This is a schematic diagram of the structure of a fully integrated multiple nucleic acid quantitative detection device of the present invention (the channel is arranged in the middle area of ​​the chamber);

[0019] Figure 3This is a schematic diagram of the initial position of the liquid in a fully integrated multiple nucleic acid quantitative detection device of the present invention (the channel is set in the top area of ​​the chamber);

[0020] Figure 4 This is a schematic diagram of the initial position of the liquid in a fully integrated multiple nucleic acid quantitative detection device of the present invention (the channel is set in the middle area of ​​the chamber);

[0021] Figure 5 Schematic diagram of uniform distribution of magnetic particles in the sample lysis chamber under the action of an external magnetic field;

[0022] Figure 6 Schematic diagram of the transport of magnetic particles under the action of an external magnetic field;

[0023] Figure 7 It is a cross-sectional view of the structure of the first reaction chamber and the first quantitative chamber;

[0024] Figure 8 Schematic diagram of the initial position of the liquid in the first reaction chamber (cross-sectional view) before and after the device is flipped over;

[0025] Figure 9 Schematic diagram of the magnetic particles and detection reagents in the reaction chamber being driven into the quantification chamber.

[0026] Among them, 1-sample lysis chamber, 2-nucleic acid cleaning chamber, 3a-first reaction chamber, 3b-second reaction chamber, 4a-first quantitative chamber, 4b-second quantitative chamber, 5-channel, 6-channel switch position, 7-tube cap, 8-partition, 9-interface, 10-lysis reagent, 11-nucleic acid cleaning reagent, 12-nucleic acid reaction reagent, 13-nucleic acid reaction reagent, 14-magnetic particles, 15-oil reagent, 16-magnet or electromagnet. DETAILED DESCRIPTION

[0027] The utility model discloses a fully integrated multiple nucleic acid quantitative detection device, such as Figure 1 and Figure 2As shown, it includes a chamber and a channel 5, wherein the chamber includes at least one sample lysis chamber 1, at least one nucleic acid cleaning chamber 2, at least two reaction chambers, and at least two quantitative chambers. The reaction chamber includes a first reaction chamber 3a and a second reaction chamber 3b, and the quantitative chamber includes a first quantitative chamber 4a and a second quantitative chamber 4b. The sample lysis chamber 1, the nucleic acid cleaning chamber 2, the first reaction chamber 3a, the second reaction chamber 3b, the first quantitative chamber 4a and the second quantitative chamber 4b are connected through the channel 5. At least one partition 8 is provided at the bottom of the sample lysis chamber 1 or the bottom of the nucleic acid cleaning chamber 2. A channel switch position 6 is provided on the channel 5, and a pressure component is provided at the channel switch position 6. Applying pressure to the channel 5 by the pressure component can prevent the reaction reagents between the chambers from mixing with each other.

[0028] The partition 8 can be one or more. For example, if one partition is set at the bottom of the sample lysis chamber 1, the bottom of the sample lysis chamber 1 is divided into two parts; if two partitions are set at the bottom of the sample lysis chamber 1, the bottom of the sample lysis chamber 1 is divided into three parts; if three partitions are set at the bottom of the sample lysis chamber 1, the bottom of the sample lysis chamber 1 is divided into four parts; and so on.

[0029] The channel 5 is arranged in the middle area of ​​the chamber or the top area of ​​the chamber.

[0030] The chamber and the channel 5 are composed of a hard material polymethyl methacrylate acrylic plate and a flexible material high-temperature resistant acrylic tape. The hard material and the flexible material can be assembled together in various ways, such as: adhesion, clamping together with a clamp, connecting with screws, etc.

[0031] The pressure component is a clamp, or the pressure component is a physical valve or a surface tension valve.

[0032] Reagent addition ports are provided on the tops of the sample lysis chamber 1 , the nucleic acid cleaning chamber 2 , the first reaction chamber 3 a and the second reaction chamber 3 b , and tube caps 7 are installed at the reagent addition ports.

[0033] The tube cap 7 is a physical sealing cover, or the tube cap 7 is a membrane that can allow air to pass through but can prevent nucleic acid molecules from passing through.

[0034] The fully integrated multiple nucleic acid quantitative detection device also includes an external pump. The first quantitative chamber 4a and the second quantitative chamber 4b are jointly provided with an interface 9. The first quantitative chamber 4a and the second quantitative chamber 4b are connected to the external pump via the interface 9.

[0035] The external pump includes a syringe pump, an air pump, and a plunger pump.

[0036] A heating device is provided below the first quantitative chamber 4a and the second quantitative chamber 4b.

[0037] The utility model also discloses a fully integrated multiple nucleic acid quantitative detection method, comprising the following steps:

[0038] Step 1: Place magnetic particles and lysis reagents in the sample lysis chamber 1, place nucleic acid cleaning reagents in the nucleic acid cleaning chamber 2, place reaction reagents in the first reaction chamber 3a, and place reaction reagents in the second reaction chamber 3b; (Oily reagents need to be added, and oily reagents must be added to the first reaction chamber 3a and the second reaction chamber 3b, while other chambers can choose to add or not add oily reagents).

[0039] Step 2: Add the sample to be tested into the sample lysis chamber 1, and then seal all chamber entrances;

[0040] Step 3: The sample lysis chamber 1 contains a lysis reagent and magnetic particles. The sample to be tested is lysed in the chamber, releasing nucleic acids, and then the magnetic particles bind to the nucleic acids. A magnet or electromagnet is placed under the sample lysis chamber 1 so that the magnetic particles in the sample lysis chamber 1 are adsorbed to the bottom of the sample lysis chamber 1.

[0041] Step 4: In the present invention, the partition 8 can be provided only at the bottom of the sample lysis chamber 1, or the partition 8 can also be provided only at the bottom of the nucleic acid cleaning chamber 2. When the partition 8 is provided at the bottom of the sample lysis chamber 1, the partition 8 does not need to be provided at the bottom of the nucleic acid cleaning chamber 2. When the partition 8 is provided at the bottom of the nucleic acid cleaning chamber 2, the partition 8 does not need to be provided at the bottom of the sample lysis chamber 1. When at least one partition 8 is provided at the bottom of the sample lysis chamber 1 and the bottom of the sample lysis chamber 1 is divided into different areas, the magnet or electromagnet first drives the magnetic particles in one area of ​​the bottom of the sample lysis chamber 1 and transfers them to the nucleic acid cleaning chamber 2 through the channel 5; when at least one partition 8 is provided at the bottom of the nucleic acid cleaning chamber 2 and the bottom of the nucleic acid cleaning chamber 2 is divided into different areas, the magnet or electromagnet first drives the magnetic particles in the sample lysis chamber 1 and transfers them to the nucleic acid cleaning chamber 2 through the channel 5 for cleaning, and then the magnet or electromagnet is placed under the nucleic acid cleaning chamber 2 so that the magnetic particles in the nucleic acid cleaning chamber 2 are adsorbed to different areas at the bottom of the nucleic acid cleaning chamber 2;

[0042] Step 5: The magnet or electromagnet drives the magnetic particles through the channel 5 into the first reaction chamber 3a, where the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagents;

[0043] Step 6: When at least one partition 8 is provided at the bottom of the sample lysis chamber 1 and the bottom of the sample lysis chamber 1 is divided into different areas, a magnet or an electromagnet is used to drive the magnetic particles in another area of ​​the bottom of the sample lysis chamber 1 into the nucleic acid cleaning chamber 2 for cleaning, and then into the second reaction chamber 3b to be evenly mixed with the reaction reagent; when at least one partition 8 is provided at the bottom of the nucleic acid cleaning chamber 2 and the bottom of the nucleic acid cleaning chamber 2 is divided into different areas, a magnet or an electromagnet is used to drive the magnetic particles in another area of ​​the bottom of the nucleic acid cleaning chamber 2 into the second reaction chamber 3b to be evenly mixed with the reaction reagent;

[0044] It should be noted that during step 6, the magnetic particles that have been adsorbed with nucleic acid inside the first reaction chamber 3a will not react with the reaction reagents in the first reaction chamber 3a.

[0045] Step 7: Flip the device 90 degrees. Driven by external force, the mixed reagent containing magnetic particles in the first reaction chamber 3a and the second reaction chamber 3b will enter the first quantitative chamber 4a and the second quantitative chamber 4b respectively. The reagents are distributed into the tiny reaction units in the first quantitative chamber 4a and the second quantitative chamber 4b for reaction.

[0046] In the present invention, an oily reagent must be added to the first reaction chamber 3a and the second reaction chamber 3b, and the other chambers may or may not be added with an oily reagent. For example, in step 1, magnetic particles and a lysis reagent are placed in the sample lysis chamber 1, a nucleic acid cleaning reagent is placed in the nucleic acid cleaning chamber 2, a reaction reagent is placed in the first reaction chamber 3a, and a reaction reagent is placed in the second reaction chamber 3b. Then, the oily reagent is added to the sample lysis chamber 1, the nucleic acid cleaning chamber 2, the first reaction chamber 3a, and the second reaction chamber 3b respectively. The density of the oily reagent is less than the density of the lysis reagent, the nucleic acid cleaning reagent, and the reaction reagent, and the oily reagent does not react with the lysis reagent, the nucleic acid cleaning reagent, and the reaction reagent. After the oily reagent is mixed with the existing reagents in each chamber, the oily reagent will float on the surface of the reaction reagent; in step 4, when the bottom of the sample lysis chamber 1 is provided with a At least one partition 8 is provided, and when the bottom of the sample lysis chamber 1 is divided into different areas, the magnet or electromagnet first drives the magnetic particles in one area of ​​the bottom of the sample lysis chamber 1 to pass through the lysis reagent and enter the oily reagent, and then transfers them to the nucleic acid cleaning chamber 2 through the channel 5; when at least one partition 8 is provided at the bottom of the nucleic acid cleaning chamber 2 and the bottom of the nucleic acid cleaning chamber 2 is divided into different areas, the magnet or electromagnet drives the magnetic particles in the sample lysis chamber 1 to pass through the lysis reagent and enter the oily reagent, and then transfers them to the nucleic acid cleaning chamber 2 through the channel 5 for cleaning, and then the magnet or electromagnet is placed under the nucleic acid cleaning chamber 2, so that the magnetic particles in the nucleic acid cleaning chamber 2 are adsorbed to different areas of the bottom of the nucleic acid cleaning chamber 2; in the step 5, the magnet or electromagnet drives the magnetic particles to pass through the nucleic acid cleaning reagent and enter the oily reagent, and then enter the first reaction chamber 3a through the channel 5, and the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagent.

[0047] In the present invention, the oily reagent can be added only to the first reaction chamber 3a and the second reaction chamber 3b. For example, in step 1, magnetic particles and a lysis reagent are placed in the sample lysis chamber 1, a nucleic acid cleaning reagent is placed in the nucleic acid cleaning chamber 2, a reaction reagent is placed in the first reaction chamber 3a, and a reaction reagent is placed in the second reaction chamber 3b. Then, the oily reagent is added to the first reaction chamber 3a and the second reaction chamber 3b respectively. The density of the oily reagent is less than the density of the reaction reagent and the oily reagent does not react with the reaction reagent. After the oily reagent is mixed with the existing reaction reagent in the first reaction chamber 3a and the second reaction chamber 3b, the oily reagent will float on the surface of the reaction reagent.

[0048] In step 3, the magnetic particles and nucleic acids in the sample lysis chamber 1 are fully mixed and contacted by repeated pressing or ultrasonic vibration.

[0049] In step 4, the magnetic particles are fully mixed and contacted with the nucleic acid cleaning reagent by repeated pressing or ultrasonic vibration to complete the cleaning.

[0050] In step 7, the external force includes capillary force, surface tension, and vacuum pressure.

[0051] The reaction reagent is a CRISPR reaction reagent, or a nucleic acid amplification reagent, or a mixed reagent of a CRISPR reaction reagent and a nucleic acid amplification reagent; the nucleic acid amplification reagent includes a PCR reagent or a constant temperature amplification reagent (such as a LAMP reaction reagent, an RPA reaction reagent, an RCA reaction reagent, etc.).

[0052] The components of the CRISPR reaction reagent include 100-500nM Cas protein, 200-2000nM crRNA, 1-20μM single-stranded signal nucleic acid, 0.1-2.5U / μL RNase inhibitor, and buffer; if the detection target is a DNA fragment, the complex of Cas protein and crRNA needs to be able to recognize the target DNA and activate the Cas protein to cut the single-stranded signal nucleic acid to obtain a detection signal; if the detection target is an RNA fragment, the complex of Cas protein and crRNA needs to be able to recognize the target RNA and activate the Cas protein to cut the single-stranded signal nucleic acid to obtain a detection signal.

[0053] The lysis reagent comprises 1-6M guanidine thiocyanate, 10-40mM ethylenediaminetetraacetic acid, 1-5% (m / v) N-lauroylsarcosine sodium salt, 20-100mM tris(hydroxymethyl)aminomethane hydrochloride, 30-80% (v / v) isopropanol, and has a pH value between 7.0-8.0.

[0054] The nucleic acid cleaning reagent comprises 50-90% (v / v) ethanol; the magnetic particles are superparamagnetic spheres with a diameter of 0.01-10 μm; and the oily reagent is paraffin oil.

[0055] The technical solution of the present invention is described below through specific embodiments:

[0056] Example 1

[0057] like Figure 3 and Figure 4As shown, the sample lysis chamber 1, nucleic acid cleaning chamber 2, first reaction chamber 3a, and second reaction chamber 3b are pre-placed with reaction reagents required for nucleic acid detection, or different reaction reagents required for nucleic acid detection are added to the sample lysis chamber 1, nucleic acid cleaning chamber 2, first reaction chamber 3a, and second reaction chamber 3b before the reaction operation. Magnetic particles 14 are also placed in the sample lysis chamber 1. Oily reagent 15 needs to be added to the first reaction chamber 3a and the second reaction chamber 3b, while the sample lysis chamber 1 and nucleic acid cleaning chamber 2 can optionally add or not add the oily reagent 15. The density of the oily reagent 15 is less than that of the reaction reagent and does not react with the reaction reagent.

[0058] like Figure 5 and Figure 6 As shown, a magnet or electromagnet 16 is provided outside the chamber. When the magnet or electromagnet 16 is located directly below the sample lysis chamber 1, the magnetic particles in the sample lysis chamber 1 are attracted to the bottom of the sample lysis chamber 1. Due to the presence of the partition 8, the magnetic particles 14 are evenly distributed to both sides of the bottom of the sample lysis chamber 1. The magnet or electromagnet 16 can control the movement of the magnetic particles 14 in the chamber into different reagent or air interfaces, thereby achieving movement between different chambers.

[0059] The tops of the sample lysis chamber 1, nucleic acid cleaning chamber 2, first reaction chamber 3a, and second reaction chamber 3b are equipped with reagent inlets, each fitted with a cap 7. After opening the cap 7, the corresponding reagent is added through the inlet. The cap 7 can be a physical seal or, if desired, contain a membrane that allows air to pass through while preventing nucleic acid molecules from passing through.

[0060] like Figure 7 、 Figure 8 and Figure 9 As shown, the bottom of the first reaction chamber 3a is connected with a channel 5, which is further communicated with the first quantitative chamber 4a. Before operating, the device is in a vertical position. After the magnetic particles 14 enter the first reaction chamber 3a and evenly mix with the nucleic acid reaction reagent 12 in the first reaction chamber 3a, the device is flipped 90 degrees. The oily reagent 15 will always float above the nucleic acid reaction reagent 12 due to its low density. The first quantitative chamber 4a is connected to an external syringe pump through an interface 9. Through the suction of the syringe pump, the nucleic acid reaction reagent 12 and the magnetic particles 14 in the first reaction chamber 3a will enter the first quantitative chamber 4a. Due to the gravity of the magnetic particles 14 themselves, the nucleic acid reaction reagent 12 will be driven to enter the micro-reaction unit in the first quantitative chamber 4a more easily. Then, the oily reagent 15 will also enter the first quantitative chamber 4a and squeeze out the redundant nucleic acid reaction reagent 12 through the interface 9. At the same time, each micro-reaction unit will be separated by the oily reagent 15 to avoid mutual interference in the reactions.

[0061] The device can ensure the sealing of the entire detection process, and the reagents in different chambers will not be mixed and contaminated. When conducting quantitative detection, on the one hand, the magnetic particles can be controlled by external magnets or electromagnets to move in different chambers to complete nucleic acid purification, avoiding pipetting operations. On the other hand, relying on the gravity of the magnetic particles themselves, it is easier to introduce reaction reagents into the tiny reaction units in the quantitative chamber, and can be used as an indicator to eliminate false positive results. In addition to sealing to prevent reagent contamination, the oily reagents in the device can also be directly introduced into the quantitative chamber to squeeze out excess reaction reagents, so that the reactions between the tiny reaction units are separated from each other to avoid interference. These features can make the operation of the device simpler and more convenient, and at the same time, the accuracy of the quantitative results is higher.

[0062] Example 2

[0063] Dual nucleic acid quantitative detection targeting the Salmonella invasion protein A (invA) gene sequence and the Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence

[0064] The structure of the device used is as follows Figure 1 and Figure 3 As shown, the channel 5 in the device is set in the top area of ​​the chamber. The materials used are polymethyl methacrylate acrylic plate and polymethyl methacrylate acrylic tape, and the oily reagent is paraffin oil. Before use, 2 microliters of magnetic particles and 300 microliters of lysis reagent are placed in the sample lysis chamber 1, 500 microliters of nucleic acid cleaning reagent is placed in the nucleic acid cleaning chamber 2, 30 microliters of CRISPR detection reagent is placed in the first reaction chamber 3a, and 30 microliters of CRISPR detection reagent is placed in the second reaction chamber 3b. Finally, paraffin oil is added so that the sample lysis chamber 1, nucleic acid cleaning chamber 2, first reaction chamber 3a, second reaction chamber 3b, and the channel 5 between them are all covered with paraffin oil.

[0065] The CRISPR detection reagent used in the present embodiment is CRISPR / Cas12a detection reagent, including Cas12a protein, crRNA, single-stranded DNA fluorescent probe (5' end modification FAM group, 3' end modification BHQ1 group), RNA enzyme inhibitor, buffer solution etc. When target DNA exists, crRNA can be paired with target DNA, and then activates the cleavage activity of Cas12a protein, cuts single-stranded DNA fluorescent probe, and produces fluorescent signal. The CRISPR detection reagent placed in the first reaction chamber 3a can detect Salmonella invasion protein A (invA) gene sequence, and the CRISPR detection reagent placed in the first reaction chamber 3b can detect Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence.

[0066] A mixed sample containing Salmonella and Vibrio parahaemolyticus was collected by centrifugation. The bacteria were then resuspended in a small amount of sterile water and added to sample lysis chamber 1. The tube cap was then secured. Channel 5 was opened to connect sample lysis chamber 1, nucleic acid cleaning chamber 2, first reaction chamber 3a, and second reaction chamber 3b.

[0067] Then, use your fingers to intermittently press the sample lysis chamber 1 to evenly mix the sample to be tested, the magnetic particles, and the lysis reagent. After incubation at room temperature for 20 minutes, place the magnet directly below the bottom of the sample lysis chamber 1. Since there is a partition 8 at the bottom of the sample lysis chamber 1, the magnetic particles in the sample lysis chamber 1 are evenly distributed to both sides of the partition 8. Then use a magnet to first transport the magnetic particles on the right side of the bottom of the sample lysis chamber 1 to the nucleic acid cleaning chamber 2, and use your fingers to intermittently press the nucleic acid cleaning chamber 2 to stir the liquid and wash for 1 minute. Finally, use a magnet to transport the magnetic particles to the first reaction chamber 3a to mix with the CRISPR detection reagent.

[0068] Next, continue to use the magnet to transfer the magnetic particles on the bottom left side of the sample lysis chamber 1 to the nucleic acid cleaning chamber 2, repeat the previous operation for cleaning, and finally transfer the magnetic particles to the second reaction chamber 3b to mix with the CRISPR detection reagent.

[0069] After the reagents in the first and second reaction chambers 3a and 3b are evenly mixed, the device is flipped 90 degrees and suction is performed at port 9 using an external syringe. The reagents (including the magnetic particles) in the first and second reaction chambers 3a and 3b are then drawn into the micro-reaction units in the first and second quantitative chambers 4a and 4b, respectively. Paraffin oil is then drawn into the first and second quantitative chambers 4a and 4b, separating the micro-reaction units and sealing the entire reaction. Suction is stopped after the paraffin oil reaches port 9.

[0070] The device was placed on a heat block at 37-42 degrees Celsius for 30 minutes. Under the irradiation of 490-nanometer wavelength excitation light, a microscope combined with smartphone photography and fluorescence signal processing and analysis software was used to obtain the quantitative results of the nucleic acid in chambers 4a and 4b, achieving dual quantitative detection.

[0071] Example 3

[0072] Dual nucleic acid quantitative detection targeting the Salmonella invasion protein A (invA) gene sequence and the Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence

[0073] The structure of the device used is as follows Figure 2As shown, channel 5 in the device is located in the middle area of ​​the chamber. The materials used are polymethyl methacrylate acrylic plate and polymethyl methacrylate acrylic tape, and the oily reagent is paraffin oil. Before use, place 2 μl of magnetic particles and 300 μl of lysis reagent in sample lysis chamber 1, 500 μl of nucleic acid cleaning reagent in nucleic acid cleaning chamber 2, 30 μl of CRISPR detection reagent in first reaction chamber 3a, and 30 μl of CRISPR detection reagent in second reaction chamber 3b. Finally, add an appropriate amount of paraffin oil only to the first reaction chamber 3a and the second reaction chamber 3b.

[0074] The CRISPR detection reagent used in the present embodiment is CRISPR / Cas12a detection reagent, including Cas12a protein, crRNA, single-stranded DNA fluorescent probe (5' end modification FAM group, 3' end modification BHQ1 group), RNA enzyme inhibitor, buffer solution etc. When target DNA exists, crRNA can be paired with target DNA, and then activates the cleavage activity of Cas12a protein, cuts single-stranded DNA fluorescent probe, and produces fluorescent signal. The CRISPR detection reagent placed in the first reaction chamber 3a can detect Salmonella invasion protein A (invA) gene sequence, and the CRISPR detection reagent placed in the second reaction chamber 3b can detect Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence.

[0075] A mixed sample containing Salmonella and Vibrio parahaemolyticus was collected by centrifugation. The bacteria were then resuspended in a small amount of sterile water and added to sample lysis chamber 1. The tube cap was then secured. Channel 5 was opened to connect sample lysis chamber 1, nucleic acid cleaning chamber 2, first reaction chamber 3a, and second reaction chamber 3b.

[0076] Then, use an electric toothbrush to shake and mix the sample to be tested, magnetic particles, and lysis reagent in the sample lysis chamber 1. After incubation at room temperature for 20 minutes, place the magnet directly below the bottom of the sample lysis chamber 1. Since there is a partition 8 at the bottom of the sample lysis chamber 1, the magnetic particles in the sample lysis chamber 1 are evenly distributed to both sides of the partition 8. Then use a magnet to first transport the magnetic particles on the right side of the bottom of the sample lysis chamber 1 to the nucleic acid cleaning chamber 2, press the nucleic acid cleaning chamber 2 intermittently with your fingers to stir the liquid, and wash for 1 minute. Finally, use a magnet to transport the magnetic particles to the first reaction chamber 3a to mix with the CRISPR detection reagent.

[0077] Next, continue to use the magnet to transfer the magnetic particles on the left side of the bottom of chamber 1 to the nucleic acid cleaning chamber 2, repeat the previous operation for cleaning, and finally transfer the magnetic particles to the second reaction chamber 3b to mix with the CRISPR detection reagent.

[0078] After the reagents in the first and second reaction chambers 3a and 3b are evenly mixed, the device is flipped 90 degrees and suction is performed at port 9 using an external syringe. The reagents (including the magnetic particles) in the first and second reaction chambers 3a and 3b are then drawn into the micro-reaction units in the first and second quantitative chambers 4a and 4b, respectively. Paraffin oil is then drawn into the first and second quantitative chambers 4a and 4b, separating the micro-reaction units and sealing the entire reaction. Suction is stopped after the paraffin oil reaches port 9.

[0079] The device was placed on a heat block at 37-42 degrees Celsius for 30 minutes. Under the irradiation of 490-nanometer wavelength excitation light, a microscope combined with smartphone photography and fluorescence signal processing and analysis software was used to obtain the quantitative results of the nucleic acid in chambers 4a and 4b, achieving dual quantitative detection.

[0080] The utility model has the advantages of simple structural design, convenient operation, easy portability, basically no need to move liquid, and can perform multiple nucleic acid quantitative analysis, including:

[0081] 1. This utility model integrates sample nucleic acid purification and multiple nucleic acid quantitative detection within a single device through a simple chamber design and layout, enabling "sample in, result out" integrated testing. This offers the advantage of a simple structural design. The entire operation process is also conducted in a sealed environment, preventing contamination from the outside world during the testing process and contamination of the reagents by the outside world.

[0082] 2. This utility model can achieve uniform mixing of magnetic particles and reagents by simply repeatedly pressing or ultrasonically vibrating the chamber. By controlling the movement of magnetic particles in the chamber through an external moving magnet or electromagnet, sample nucleic acid purification and mixing with nucleic acid detection reagents can be completed. It has the advantages of easy operation, no need to move liquid, and no need to consider the balance of internal and external air pressure.

[0083] 3. This utility model mixes magnetic particles with nucleic acid detection reagents in a reaction chamber and then injects them into a micro-reaction unit in a quantitative chamber for quantitative detection. The magnetic particles carry the reagents on their surfaces, allowing them to more easily enter the micro-reaction units due to their own gravity. Furthermore, observing whether the magnetic particles have entered the micro-reaction units can be used to indicate whether the reagents have entered the micro-reaction units, thereby eliminating false negative results and providing high accuracy.

[0084] 4. After the magnetic particles are mixed with the nucleic acid detection reagent in the reaction chamber, the device is simply flipped 90 degrees. Driven by external forces (such as capillary force, surface tension, vacuum pressure, etc.), the reaction reagents can be driven into the tiny reaction units in the quantitative chamber. At the same time, the oily reagents floating above the reaction reagents in the reaction chamber will also enter the quantitative chamber. On the one hand, the excess reaction reagents in the quantitative chamber can be squeezed out, and on the other hand, the reactions between each tiny reaction unit can be separated from each other to avoid mutual interference.

[0085] 5. The device of this utility model has flexible configuration. The first and second reaction chambers can be used to house nucleic acid amplification reagents (including variable temperature amplification and constant temperature amplification), CRISPR reaction reagents, and even a mixture of CRISPR reaction reagents and nucleic acid amplification reaction reagents, enabling multiple quantitative analysis of nucleic acids. The device can also be used for other molecular biology or immunoassay experiments with similar operational requirements.

[0086] 6. The device used in the utility model can be manufactured using low-cost materials, the operation process is simple, and the cost of the reagents used is comparable to the cost of reagents for normal related reactions.

[0087] The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention cannot be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A fully integrated multiplex nucleic acid quantitative detection device, characterized in that: The invention comprises a chamber and a channel (5), wherein the chamber comprises at least one sample lysis chamber (1), at least one nucleic acid cleaning chamber (2), at least two reaction chambers, and at least two quantitative chambers. The chambers are connected through the channel (5), at least one partition (8) is provided at the bottom of the sample lysis chamber (1) or the bottom of the nucleic acid cleaning chamber (2), and the quantitative chamber contains a plurality of micro-reaction units; a channel switch position (6) is provided on the channel (5), and a pressure component is provided at the channel switch position (6). The pressure component applies pressure to the channel (5) to prevent the reaction reagents between the chambers from mixing with each other.

2. The fully integrated multiplex nucleic acid quantitative detection device according to claim 1, characterized in that: The channel (5) is arranged in the middle area of ​​the chamber or the top area of ​​the chamber.

3. The fully integrated multiplex nucleic acid quantitative detection device according to claim 1, characterized in that: The chamber and the channel (5) are composed of a hard material and a flexible material, and the hard material and the flexible material are assembled together.

4. The fully integrated multiplex nucleic acid quantitative detection device according to claim 1, characterized in that: The pressure component is a clamp, or the pressure component is a physical valve or a surface tension valve.

5. The fully integrated multiplex nucleic acid quantitative detection device according to claim 1, characterized in that: The reaction chamber comprises a first reaction chamber (3a) and a second reaction chamber (3b); the sample lysis chamber (1), the nucleic acid cleaning chamber (2), the first reaction chamber (3a), and the second reaction chamber (3b) are provided with reagent addition ports at the top, and tube caps (7) are installed at the reagent addition ports.

6. The fully integrated multiplex nucleic acid quantitative detection device according to claim 5, characterized in that: The tube cap (7) is a physical sealing cover, or the tube cap (7) is a membrane that can pass air but can prevent nucleic acid molecules from passing through.

7. The fully integrated multiplex nucleic acid quantitative detection device according to claim 1, characterized in that: The fully integrated multiple nucleic acid quantitative detection device further comprises an external pump, the quantitative chamber comprising a first quantitative chamber (4a) and a second quantitative chamber (4b), the first quantitative chamber (4a) and the second quantitative chamber (4b) being provided with a common interface (9), and the first quantitative chamber (4a) and the second quantitative chamber (4b) being connected to the external pump via the interface (9).

8. The fully integrated multiplex nucleic acid quantitative detection device according to claim 7, characterized in that: The external pump includes a syringe pump, an air pump, and a plunger pump.

9. The fully integrated multiplex nucleic acid quantitative detection device according to claim 7, characterized in that: A heating device is provided below the first quantitative chamber (4a) and the second quantitative chamber (4b).

10. The fully integrated multiplex nucleic acid quantitative detection device according to claim 3, characterized in that: The hard material is a polymethyl methacrylate acrylic plate, and the flexible material is a high-temperature resistant acrylic tape.

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