Fully integrated multiplex nucleic acid quantitative detection method

Abstract

The invention provides a fully-integrated multiplex nucleic acid quantitative detection method. Magnetic particles, a nucleic acid cracking reagent, a nucleic acid cleaning reagent, a nucleic acid reaction reagent and an oily reagent are added into corresponding chambers in a device. A to-be-detected sample is added into the sample cracking chamber, and the magnetic particles and the reagent are uniformly mixed by pressing or vibrating the chamber. Then, the magnetic particles in the chamber are driven by an external magnet or electromagnet to sequentially enter the nucleic acid cleaning chamber and the reaction chamber through the channel, and pipetting is not needed. A partition plate is arranged in the sample cracking chamber or the nucleic acid cleaning chamber, and a magnet or an electromagnet is arranged under the chamber provided with the partition plate, so that magnetic particles can be uniformly divided into a plurality of parts. The magnetic particles which are divided into a plurality of parts can enter different reaction chambers respectively to be mixed with nucleic acid reaction reagents. Finally, the device is turned over by 90 degrees, the mixture of the magnetic particles and the nucleic acid reaction reagent and the oily reagent sequentially enter the tiny reaction units in the quantitative chamber, and quantitative detection is carried out. The method has the beneficial effects that the whole operation process is carried out in a closed state, so that the pollution to the outside or the pollution to a reagent caused by the outside in the detection process can be avoided; operation is convenient, liquid driving is little, and internal and external air pressure balance does not need to be considered.

Description

Technical Field

[0001] The present invention relates to the field of detection and analysis technology, and in particular to a fully integrated multiple nucleic acid quantitative detection method. 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 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 affect the experimental results during the operation. 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 drives the liquid to flow in a microchannel through rotary centrifugation, and has high requirements for the speed control of the supporting instrument. Chinese patent CN 114292734 B relates to a full-process integrated droplet digital PCR chip, preparation method and application, which uses droplets combined with PCR for integrated quantitative detection, requires the help of temperature control instruments, the uniformity of droplet size generation is difficult to control, the droplets are not very stable under high temperature conditions, and multiple analyses cannot be performed. Chinese patent CN 116103139 B relates to a fully enclosed fully integrated nucleic acid detection microfluidic chip, which requires special instruments, has a complex structural design, high cost, and cannot perform multiple quantitative detection. Chinese patent CN 107129930 B relates to a fully integrated nucleic acid detection microfluidic chip and its use method, which uses many pump valve structures on the chip, resulting in complex chip manufacturing and high cost. Chinese patent CN 114931986 B relates to a nucleic acid detection microfluidic device and its nucleic acid detection method, the chip structure design is also complex, and can only perform qualitative analysis of nucleic acids. At present, some integrated nucleic acid detection systems have also been launched on the market, but most devices drive liquids to work, the structure is relatively complex, and the supporting instruments are expensive.

[0003] The CRISPR / Cas system is a type of adaptive immune system in bacteria and archaea, mainly composed of Cas proteins and corresponding crRNA. With the discovery of the bypass cleavage effect of some Cas proteins, some researchers have established a new nucleic acid detection platform based on the CRISPR / Cas system. When the target nucleic acid is present, under the guidance of crRNA, the Cas protein and crRNA complex accurately recognize and bind to the target nucleic acid, thereby stimulating the bypass cleavage activity of the Cas protein, cutting 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, which can only perform qualitative detection of nucleic acids and the operation process requires multiple liquid transfers, which is complicated. Chinese patent CN 108823291 B discloses a specific nucleic acid fragment quantitative detection method based on CRISPR technology, which cannot complete the sample nucleic acid pretreatment and requires a droplet generation device, which increases the complexity of the device.

[0004] Nucleic acid quantitative detection is extremely important, especially in the field 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 corresponding treatment plans. Nucleic acid quantitative detection of pathogenic microorganisms in food is one of the important indicators for measuring food hygiene quality and can make a correct evaluation of 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 capable of multiple quantitative analysis. Moreover, the device can be combined with the CRISPR / Cas system to carry out multiple nucleic acid quantitative analysis. Summary of the invention

[0006] The present invention provides a fully integrated multiple nucleic acid quantitative detection method, comprising the following steps: Step 1: placing magnetic particles and a lysis reagent in a sample lysis chamber, placing a nucleic acid cleaning reagent in a nucleic acid cleaning chamber, placing a reaction reagent in a first reaction chamber, and placing a reaction reagent in a second reaction chamber;

[0007] Step 2: Add the sample to be tested into the sample lysis chamber, and then seal all chamber entrances;

[0008] Step 3: The sample lysis chamber contains a lysis reagent and magnetic particles, the sample to be tested is lysed and nucleic acids are released in the chamber, and then the magnetic particles are combined with the nucleic acids; a magnet or an electromagnet is placed under the sample lysis chamber so that the magnetic particles in the sample lysis chamber are adsorbed to the bottom of the sample lysis chamber;

[0009] Step 4: When at least one partition is provided at the bottom of the sample lysis chamber and the bottom of the sample lysis chamber 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 and transfers them to the nucleic acid cleaning chamber through the channel; when at least one partition is provided at the bottom of the nucleic acid cleaning chamber and the bottom of the nucleic acid cleaning chamber is divided into different areas, the magnet or electromagnet drives the magnetic particles in the sample lysis chamber and transfers them to the nucleic acid cleaning chamber through the channel for cleaning, and then places the magnet or electromagnet under the nucleic acid cleaning chamber so that the magnetic particles in the nucleic acid cleaning chamber are adsorbed to different areas at the bottom of the nucleic acid cleaning chamber;

[0010] Step 5: The magnet or electromagnet drives the magnetic particles through the channel into the first reaction chamber, and the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagents;

[0011] Step 6: When at least one partition is provided at the bottom of the sample lysis chamber and the bottom of the sample lysis chamber 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 into the nucleic acid cleaning chamber for cleaning, and then enter the second reaction chamber to be evenly mixed with the reaction reagent; When at least one partition is provided at the bottom of the nucleic acid cleaning chamber and the bottom of the nucleic acid cleaning chamber 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 into the second reaction chamber to be evenly mixed with the reaction reagent;

[0012] When performing step 6, the magnetic particles on which the nucleic acid has been adsorbed in the first reaction chamber will not react with the reaction reagent in the first reaction chamber;

[0013] Step 7: Flip the device 90 degrees. Driven by external force, the mixed reagent containing magnetic particles in the first reaction chamber and the second reaction chamber will enter the first quantitative chamber and the second quantitative chamber respectively, and the reagents will be distributed into the tiny reaction units in the first quantitative chamber and the second quantitative chamber for reaction.

[0014] As a further improvement of the present invention, in the step 1, magnetic particles and a lysis reagent are placed in the sample lysis chamber, a nucleic acid cleaning reagent is placed in the nucleic acid cleaning chamber, a reaction reagent is placed in the first reaction chamber, and a reaction reagent is placed in the second reaction chamber, and then an oily reagent is added to the sample lysis chamber, the nucleic acid cleaning chamber, the first reaction chamber, and the second reaction chamber, 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, and after the oily reagent is mixed with the existing reagent in each chamber, the oily reagent will float on the surface of the reagent;

[0015] In step 4, when at least one partition is provided at the bottom of the sample lysis chamber and the bottom of the sample lysis chamber 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 to pass through the lysis reagent and enter the oily reagent, and then transfers them to the nucleic acid cleaning chamber through the channel; when at least one partition is provided at the bottom of the nucleic acid cleaning chamber and the bottom of the nucleic acid cleaning chamber is divided into different areas, the magnet or electromagnet drives the magnetic particles in the sample lysis chamber to pass through the lysis reagent and enter the oily reagent, and then transfers them to the nucleic acid cleaning chamber through the channel for cleaning, and then the magnet or electromagnet is placed under the nucleic acid cleaning chamber, so that the magnetic particles in the nucleic acid cleaning chamber are adsorbed to different areas at the bottom of the nucleic acid cleaning chamber;

[0016] In step 5, the magnet or electromagnet drives the magnetic particles to pass through the nucleic acid cleaning reagent into the oily reagent, and then enter the first reaction chamber through the channel, and the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagent.

[0017] As a further improvement of the present invention, in step 1, magnetic particles and a lysis reagent are placed in the sample lysis chamber, a nucleic acid cleaning reagent is placed in the nucleic acid cleaning chamber, a reaction reagent is placed in the first reaction chamber, and a reaction reagent is placed in the second reaction chamber, and then an oily reagent is added to the first reaction chamber and the second reaction chamber 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, and after the oily reagent is mixed with the existing reaction reagent in the first reaction chamber and the second reaction chamber, the oily reagent will float on the surface of the reaction reagent.

[0018] As a further improvement of the present invention, in step 3, the magnetic particles and nucleic acids in the sample lysis chamber are fully mixed and contacted by repeated pressing or ultrasonic vibration.

[0019] As a further improvement of the present invention, in step 4, the magnetic particles are fully mixed and contacted with the nucleic acid cleaning reagent in the nucleic acid cleaning chamber by repeated pressing or ultrasonic vibration to complete the cleaning.

[0020] As a further improvement of the present invention, in step 7, the external force includes capillary force, surface tension, and vacuum pressure.

[0021] As a further improvement of the present invention, 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.

[0022] As a further improvement of the present invention, the CRISPR reaction reagent components 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.

[0023] As a further improvement of the present invention, the lysis reagent components include 1-6M guanidine thiocyanate, 10-40mM ethylenediaminetetraacetic acid, 1-5% (m / v) N-lauroylsarcosine sodium salt, 20-100mM tris(hydroxymethylaminomethane) hydrochloride, 30-80% (v / v) isopropanol, and the pH value is between 7.0-8.0.

[0024] As a further improvement of the present invention, 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.

[0025] The beneficial effects of the present invention are as follows: the entire operation process of the present invention is also carried out in a closed state, which can avoid contamination to the outside world during the detection process or contamination of the reagent by the outside world; the present invention is easy to operate, has less liquid drive, and does not need to consider the balance of internal and external air pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It 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);

[0027] 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);

[0028] Figure 3 A schematic diagram of the initial position of liquid in a fully integrated multiple nucleic acid quantitative detection device of the present invention (the channel is arranged in the top area of ​​the chamber);

[0029] 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 arranged in the middle area of ​​the chamber);

[0030] Figure 5 Schematic diagram of uniform distribution of magnetic particles in the sample lysis chamber under the action of an external magnetic field;

[0031] Figure 6 Schematic diagram of the transport of magnetic particles under the action of an external magnetic field;

[0032] Figure 7 It is a cross-sectional view of the structure of the first reaction chamber and the first quantitative chamber;

[0033] Figure 8 A schematic diagram of the initial position of the liquid in the first reaction chamber (cross-sectional view) before and after the device is flipped;

[0034] Fig. 9 Schematic diagram of magnetic particles and detection reagents in the reaction chamber being driven into the quantification chamber.

[0035] 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-oily reagent, 16-magnet or electromagnet. DETAILED DESCRIPTION

[0036] The present invention discloses a fully integrated multiple nucleic acid quantitative detection device, such as Figure 1 and Figure 2 As 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 arranged at the bottom of the sample lysis chamber 1 or the bottom of the nucleic acid cleaning chamber 2. There is a channel switch position 6 on the channel 5, and a pressure component is arranged 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.

[0037] The partition 8 can be one or more. For example, when 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; when 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; when 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.

[0038] The channel 5 is arranged in the middle area of ​​the chamber or in the top area of ​​the chamber.

[0039] 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 by a clamp, connecting by screws, etc.

[0040] The pressure component is a clamp, or the pressure component is a physical valve or a surface tension valve.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The external pump includes a syringe pump, an air pump, and a plunger pump.

[0045] A heating device is provided below the first quantitative chamber 4a and the second quantitative chamber 4b.

[0046] The present invention also discloses a fully integrated multiple nucleic acid quantitative detection method, comprising the following steps:

[0047] 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;

[0048] Step 2: Add the sample to be tested into the sample lysis chamber 1, and then seal all chamber entrances;

[0049] Step 3: The sample lysis chamber 1 contains a lysis reagent and magnetic particles, the sample to be tested is lysed and nucleic acids are released in the chamber, and then the magnetic particles are combined with the nucleic acids; a magnet or an 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;

[0050] Step 4: In the present invention, the partition 8 can be only arranged at the bottom of the sample lysis chamber 1, or the partition 8 can also be only arranged at the bottom of the nucleic acid cleaning chamber 2. When the partition 8 is arranged at the bottom of the sample lysis chamber 1, the partition 8 does not need to be arranged at the bottom of the nucleic acid cleaning chamber 2. When the partition 8 is arranged at the bottom of the nucleic acid cleaning chamber 2, the partition 8 does not need to be arranged at the bottom of the sample lysis chamber 1. When at least one partition 8 is arranged 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 to be transferred to the nucleic acid cleaning chamber 2 through the channel 5; when at least one partition 8 is arranged 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 be transferred 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;

[0051] Step 5: The magnet or electromagnet drives the magnetic particles to enter the first reaction chamber 3a through the channel 5, and the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagents;

[0052] 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 enter 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;

[0053] It should be noted here that when performing step 6, the magnetic particles that have been adsorbed with nucleic acid inside the first reaction chamber 3a will not react with the reaction reagent in the first reaction chamber 3a;

[0054] 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, and the reagents will be distributed into the tiny reaction units in the first quantitative chamber 4a and the second quantitative chamber 4b for reaction.

[0055] 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 set 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.

[0056] In the present invention, the oily reagent can be added only in the first reaction chamber 3a and the second reaction chamber 3b. For example, in step 1, magnetic particles and lysis reagents are placed in the sample lysis chamber 1, nucleic acid cleaning reagents are placed in the nucleic acid cleaning chamber 2, reaction reagents are placed in the first reaction chamber 3a, and reaction reagents are 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.

[0057] 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.

[0058] 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.

[0059] In step 7, the external force includes capillary force, surface tension, and vacuum pressure.

[0060] 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.).

[0061] 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.

[0062] The lysis reagent comprises 1-6M guanidine thiocyanate, 10-40mM ethylenediaminetetraacetic acid, 1-5% (m / v) N-lauroylsarcosine sodium salt, 20-100mM tris(hydroxymethylaminomethane) hydrochloride, 30-80% (v / v) isopropanol, and the pH value is between 7.0-8.0.

[0063] 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.

[0064] The technical solution of the present invention is described below through specific embodiments:

[0065] Example 1

[0066] like Figure 3 and Figure 4As shown, the sample lysis chamber 1, the nucleic acid cleaning chamber 2, the first reaction chamber 3a, and the 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, the nucleic acid cleaning chamber 2, the first reaction chamber 3a, and the second reaction chamber 3b before the reaction operation. Magnetic particles 14 are also placed in the sample lysis chamber 1, and oily reagents 15 need to be added to the first reaction chamber 3a and the second reaction chamber 3b, while the sample lysis chamber 1 and the nucleic acid cleaning chamber 2 can choose to add or not add oily reagents 15. The density of the oily reagent 15 is less than that of the reaction reagent and does not react with the reaction reagent.

[0067] 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 magnetic particles 14 in the chamber to move into different reagents or air interfaces, thereby moving between different chambers.

[0068] The tops of 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, and tube caps 7 are installed at the reagent addition ports. After opening the tube caps 7, the corresponding reagents are added from the reagent addition ports. The tube caps 7 can be physical sealing covers, or they can be provided with a diaphragm that can both allow air to pass through and prevent nucleic acid molecules from passing through as required.

[0069] like Figure 7 , Figure 8 and Fig. 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 operation, the device is in a vertical placement state. 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, and 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 injection pump through an interface 9. Through the suction of the injection 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, and 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, and each micro-reaction unit will be separated by the oily reagent 15 to avoid mutual interference in the reaction.

[0070] The device can ensure the sealing of the entire detection process, and the reagents in different chambers will not mix and cause contamination. When performing 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 and avoid pipetting operations. On the other hand, relying on the gravity of the magnetic particles themselves, it is easier to introduce the 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 device easier and more convenient to operate, and at the same time, the accuracy of the quantitative results is higher.

[0071] Example 2

[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 1 and Figure 3 As shown, the channel 5 in the device is arranged 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, the nucleic acid cleaning chamber 2, the first reaction chamber 3a, the second reaction chamber 3b and the channel 5 between them are covered with paraffin oil.

[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, etc. When the target DNA exists, crRNA will be paired with the target DNA, and then activate the cutting activity of Cas12a protein, cut the single-stranded DNA fluorescent probe, and generate a fluorescent signal. The CRISPR detection reagent placed in the first reaction chamber 3a can detect the Salmonella invasion protein A (invA) gene sequence, and the CRISPR detection reagent placed in the first reaction chamber 3b can detect the Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence.

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

[0076] Then, intermittently press the sample lysis chamber 1 with your fingers to evenly mix the sample to be tested, the magnetic particles, and the lysis reagent. After incubating 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 intermittently press the nucleic acid cleaning chamber 2 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 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.

[0078] After the reagents in the first reaction chamber 3a and the second reaction chamber 3b are mixed evenly, the device is turned over 90 degrees, and suction is performed at the interface 9 using an external syringe. At this time, the reagents (including magnetic particles) in the first reaction chamber 3a and the second reaction chamber 3b are respectively sucked into the micro-reaction units in the first quantitative chamber 4a and the second quantitative chamber 4b, and the paraffin oil is finally sucked into the first quantitative chamber 4a and the second quantitative chamber 4b to separate the micro-reaction units and seal the entire reaction. After the paraffin oil reaches the interface 9, the suction is stopped.

[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, thus achieving dual quantitative detection.

[0080] Example 3

[0081] Dual nucleic acid quantitative detection targeting the Salmonella invasion protein A (invA) gene sequence and the Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence

[0082] The structure of the device used is as follows Figure 2As shown, the channel 5 in the device is arranged 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, 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, only an appropriate amount of paraffin oil is added to the first reaction chamber 3a and the second reaction chamber 3b.

[0083] 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, etc. When the target DNA exists, crRNA will be paired with the target DNA, and then activate the cutting activity of Cas12a protein, cut the single-stranded DNA fluorescent probe, and generate a fluorescent signal. The CRISPR detection reagent placed in the first reaction chamber 3a can detect the Salmonella invasion protein A (invA) gene sequence, and the CRISPR detection reagent placed in the second reaction chamber 3b can detect the Vibrio parahaemolyticus thermolabile hemolysin (tlh) gene sequence.

[0084] The mixed sample containing Salmonella and Vibrio parahaemolyticus is collected by centrifugation, and then the bacteria are resuspended in a small amount of sterile water and added to the sample lysis chamber 1, and the tube cap is covered. The channel 5 is opened to connect the sample lysis chamber 1, the nucleic acid cleaning chamber 2, the first reaction chamber 3a, and the second reaction chamber 3b.

[0085] 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, and intermittently press the nucleic acid cleaning chamber 2 with your fingers to stir the liquid and clean for 1 minute. Finally, use a magnet to transport the magnetic particles to the first reaction chamber 3a and mix them with the CRISPR detection reagent.

[0086] Next, the magnet is used to transport the magnetic particles on the left side of the bottom of chamber 1 to the nucleic acid cleaning chamber 2, and the previous operation is repeated for cleaning. Finally, the magnetic particles are transported to the second reaction chamber 3b and mixed with the CRISPR detection reagent.

[0087] After the reagents in the first reaction chamber 3a and the second reaction chamber 3b are mixed evenly, the device is turned over 90 degrees, and suction is performed at the interface 9 using an external syringe. At this time, the reagents (including magnetic particles) in the first reaction chamber 3a and the second reaction chamber 3b are respectively sucked into the micro-reaction units in the first quantitative chamber 4a and the second quantitative chamber 4b, and the paraffin oil is finally sucked into the first quantitative chamber 4a and the second quantitative chamber 4b to separate the micro-reaction units and seal the entire reaction. After the paraffin oil reaches the interface 9, the suction is stopped.

[0088] 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, thus achieving dual quantitative detection.

[0089] The present invention has the advantages of simple structural design, convenient operation, easy to carry, basically no need to move liquid, and can perform multiple nucleic acid quantitative analysis, specifically including:

[0090] 1. The invention integrates sample nucleic acid purification and multiple nucleic acid quantitative detection in one device through a simple chamber design and layout, and performs integrated detection of "sample in, result out", which has the advantage of simple structural design. The entire operation process is also carried out in a closed state, which can avoid contamination of the outside world during the detection process or contamination of the reagents by the outside world.

[0091] 2. This invention can achieve uniform mixing of magnetic particles and reagents through simple repeated pressing or ultrasonic vibration of the chamber. The movement of magnetic particles in the chamber can be controlled by an external moving magnet or electromagnet to complete sample nucleic acid purification and mixing with nucleic acid detection reagents. 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.

[0092] 3. After the magnetic particles and nucleic acid detection reagents are mixed in the reaction chamber, they are injected into the micro-reaction unit in the quantitative chamber for quantitative detection. On the one hand, the magnetic particles carry reaction reagents on their surface, and the reaction reagents can enter the micro-reaction unit more easily by relying on the gravity of the magnetic particles themselves; on the other hand, by observing whether the magnetic particles have entered the micro-reaction unit, it can be used to indicate whether the reaction reagents have entered the micro-reaction unit, thereby eliminating false negative results, which has the advantage of high accuracy.

[0093] 4. After the magnetic particles are mixed with the nucleic acid detection reagent in the reaction chamber, the invention only needs to flip the device 90 degrees. Under the drive of external forces (such as capillary force, surface tension, vacuum pressure, etc.), the reaction reagent can be driven into the tiny reaction unit in the quantitative chamber. At the same time, the oily reagent floating above the reaction reagent in the reaction chamber will also enter the quantitative chamber. On the one hand, the excess reaction reagent 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.

[0094] 5. The device of the invention has flexible configuration. The first reaction chamber and the second reaction chamber can be used to place nucleic acid amplification reaction reagents (including variable temperature amplification and constant temperature amplification), CRISPR reaction reagents, and mixed reagents of CRISPR reaction reagents and nucleic acid amplification reaction reagents to achieve multiple quantitative analysis of nucleic acids. At the same time, the device can also be used for other molecular biology detection or immune detection experiments with similar operation requirements.

[0095] 6. The device used in the invention 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 the reagents used for normal related reactions.

[0096] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A fully integrated multiplex nucleic acid quantitative detection method, characterized in that: The steps include: Step 1: placing magnetic particles and a lysis reagent in the sample lysis chamber (1), placing a nucleic acid cleaning reagent in the nucleic acid cleaning chamber (2), placing a reaction reagent in the first reaction chamber (3a), and placing a reaction reagent in the second reaction chamber (3b); Step 2: Add the sample to be tested into the sample lysis chamber (1), and then seal all chamber entrances; Step 3: The sample lysis chamber (1) contains a lysis reagent and magnetic particles, the sample to be tested is lysed in the chamber, the nucleic acid is released, and then the magnetic particles are combined with the nucleic acid; a magnet or an 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); Step 4: 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) to be transferred 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 be transferred 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); Step 5: The magnet or electromagnet drives the magnetic particles to enter the first reaction chamber (3a) through the channel (5), and the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagent; 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; When performing step 6, the magnetic particles that have been adsorbed with nucleic acid in the first reaction chamber (3a) will not react with the reaction reagent in the first reaction chamber (3a); 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, and the reagents will be distributed into the tiny reaction units in the first quantitative chamber (4a) and the second quantitative chamber (4b) for reaction.

2. The fully integrated multiple nucleic acid quantitative detection method according to claim 1, characterized in that: In the 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, an 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 floats on the surface of the reagents. In step 4, 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) 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 below 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); In step 5, the magnet or electromagnet drives the magnetic particles to pass through the nucleic acid cleaning reagent into the oily reagent, and enter the first reaction chamber (3a) through the channel (5), and the magnetic particles adsorbed with nucleic acid are mixed with the reaction reagent.

3. The fully integrated multiplex nucleic acid quantitative detection method according to claim 1, characterized in that: In the 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, an 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 floats on the surface of the reaction reagent.

4. The fully integrated multiplex nucleic acid quantitative detection method according to claim 1, characterized in that: 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.

5. The fully integrated multiplex nucleic acid quantitative detection method according to claim 1, characterized in that: In step 4, the magnetic particles are fully mixed and contacted with the nucleic acid cleaning reagent in the nucleic acid cleaning chamber (2) by repeated pressing or ultrasonic vibration to complete the cleaning.

6. The fully integrated multiplex nucleic acid quantitative detection method according to claim 1, characterized in that: In step 7, the external force includes capillary force, surface tension, and vacuum pressure.

7. The fully integrated multiplex nucleic acid quantitative detection method according to claim 1, characterized in that: 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.

8. The fully integrated multiplex nucleic acid quantitative detection method according to claim 7, characterized in that: 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.

9. The fully integrated multiplex nucleic acid quantitative detection method according to claim 1, characterized in that: The lysis reagent comprises 1-6M guanidine thiocyanate, 10-40mM ethylenediaminetetraacetic acid, 1-5% (m / v) N-lauroylsarcosine sodium salt, 20-100mM tris(hydroxymethylaminomethane) hydrochloride, 30-80% (v / v) isopropanol, and the pH value is between 7.0-8.

0.

10. The fully integrated multiplex nucleic acid quantitative detection method according to claim 2 or 3, characterized in that: 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.

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