CN112708532B - Nucleic acid detection equipment capable of continuously working - Google Patents

Abstract

The present invention relates to a nucleic acid detecting apparatus which can operate continuously, comprising: the nucleic acid amplification reaction module comprises a micro-fluidic chip, and a sample mixed solution to be detected can be injected into a flow channel and an amplification reaction chamber of the micro-fluidic chip and carries out nucleic acid amplification reaction in the amplification reaction chamber; the fluorescence detection device is suitable for detecting a fluorescence signal of the amplification reaction chamber so as to obtain the amount of an amplification product in the amplification reaction chamber in real time according to the fluorescence signal; and a residue removing module which comprises a high-temperature fluid cleaning module and is used for injecting fluid with the temperature of more than 200 ℃ into the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber when the detection result of the nucleic acid detection equipment is positive so as to remove positive residues. The invention can automatically remove the positive residues in the equipment, thereby avoiding the occurrence of false positives in subsequent samples; meanwhile, the invention can obviously reduce the use cost.

Description

Nucleic acid detection equipment capable of continuously working

Technical Field

The invention relates to the technical field of nucleic acid detection, aerosol detection and the like, in particular to nucleic acid detection equipment capable of working continuously.

Background

There are a lot of cases showing that new coronary pneumonia has a long latent period compared with SARS epidemic situation, and even asymptomatic infectors of new coronary pneumonia can become virus propagators, and the relationship between fever symptoms of virus infected propagators and the ability of infecting others is neither sufficient nor necessary, which aggravates the difficulty of accurately controlling virus propagation. People urgently expect a solution of non-contact virus monitoring to quickly and accurately identify viruses, provide help for epidemic situation prevention and control, further recover production and living order and reduce economic and social losses.

Nucleic acid detection is currently the main method used to detect new corona virus and to confirm the diagnosis of new corona pneumonitis. Currently, the national Weijian Commission recommends two types of nucleic acid detection methods for identifying new coronavirus, one is the sequencing method and the other is the RT-PCR method. Sequencing generally takes 1-2 days, while RT-PCR generally takes no less than 3-6 hours. It can be seen that the current nucleic acid detection scheme is too long in time consumption and still difficult to meet the requirement of accurate epidemic prevention and control. Moreover, these two methods currently in use require medical personnel to sample the patient or suspected patient, and the medical personnel are at great risk of infection during the sampling process. These existing problems will be described in more detail below.

Sequencing is to perform genome sequencing on nucleic acid extracted from a sample and identify whether the sample contains virus particles or not according to a sequencing result. Compared with RT-PCR, sequencing method usually needs longer time, and is difficult to realize the rapid detection of new coronavirus. The RT-PCR is called Reverse Transcription-Polymerase Chain Reaction. RT refers to reverse transcription, also known as reverse transcription. PCR refers to polymerase chain amplification. The RT-PCR method is a technique in which reverse transcription of RNA and polymerase chain amplification of cDNA (i.e., PCR) are combined. Firstly, cDNA is synthesized from RNA under the action of reverse transcriptase, and then the target fragment is amplified and synthesized under the action of DNA polymerase by taking the cDNA as a template. RT-PCR technology is sensitive and widely used, and is currently used for detecting gene expression level in cells, RNA virus content in cells and directly cloning cDNA sequences of specific genes. The new coronavirus belongs to RNA virus, so that the RT-PCR method can be used for detecting the new coronavirus.

Further, the fluorescent quantitative PCR is a method for measuring the total amount of products after the polymerase chain reaction (i.e., PCR) cycle in a DNA amplification reaction using a fluorescent chemical. Furthermore, in the PCR amplification process, the PCR process is detected in real time through a fluorescent signal, so that the real-time fluorescent quantitative PCR can be realized. In the exponential phase of PCR amplification, the Ct value of the template (Ct is totally called Cycle threshold, and Ct value can be translated into Cycle threshold) and the initial copy number of the template have a linear relation, so that the method can be used as a basis for quantitative detection of viruses. The principle is that a fluorescent group is added into a PCR reaction system, the whole PCR process is monitored in real time by using fluorescent signal accumulation, and finally, an unknown template (such as virus nucleic acid) is quantitatively analyzed through a detected real-time amplification curve.

In addition to the nucleic acid detection methods described above, the colloidal gold method is also used for virus detection. Colloidal gold is a common labeling technology, and is an immune labeling technology which applies colloidal gold as a tracer marker to antigen and antibody. The applications of colloidal gold methods in medical tests are mainly immunochromatography and rapid immunogold filtration, which are used for detecting HBsAg, HCG, anti-double-stranded DNA antibodies, and the like. Although the colloidal gold method can be used for virus detection, the sensitivity of the method is different from that of nucleic acid detection methods such as RT-PCR. For the new coronary pneumonia, the existing colloidal gold method is mainly suitable for detecting viruses of patients with 3-7 days of attack, and when the virus content of a sample is low (such as a sample of a patient with latent, mild or even asymptomatic infection), a false negative problem can exist. Therefore, the colloidal gold method is difficult to be used for rapid virus detection with high sensitivity. In addition, the colloidal gold method is often based on gel electrophoresis technology to analyze and identify DNA molecules. Various factors such as the relative mass (molecular weight) of the DNA molecule, the concentration of the gel (e.g., agarose), the conformation of the DNA molecule (e.g., different conformations such as linear, open-loop, and supercoiled), etc., may influence the results of the identification. For example, linear, open-loop and supercoiled DNA of the same molecular weight move at different speeds in agarose gel, which results in the possibility that DNA molecules of different molecular weights and conformations move at the same speed, so that the DNA molecules of different molecular weights and conformations are exactly on the same spectral line of the test paper. That is, if a molecule exists in the sample, the molecular weight and conformation of which are such that the electrophoretic moving speed of the molecule is exactly the same as that of the target DNA molecule, the corresponding line on the test paper may show positive, thereby causing a problem of false positive. The colloidal gold method is still insufficient in reliability as compared with the RT-PCR method.

On the other hand, in the current epidemic prevention and control, reducing the infection risk of medical staff is also one of the important problems to be solved urgently. The existing new coronavirus PCR detection equipment needs to be used for sampling patients or suspected patients by medical staff, such as nasal swabs (nasopharyngeal swabs), pharyngeal swabs (oropharyngeal swabs), aspirates and the like. These sampling procedures are prone to patient discomfort, leading to difficult sampling. If the sampling time is too long, the medical staff will be exposed to a great risk of infection. Therefore, a PCR detection device capable of self-help detection by patients is urgently expected, so that the exposure risk of medical staff is reduced. Some existing influenza virus detection devices can be partially automated, but they often perform virus detection based on test tubes, disposable biochips or other disposable containers, which results in many links in the virus detection process still being unable to be automated, and these links which are unable to be automated often have to be intervened by professionals.

Furthermore, there is evidence that new coronaviruses can be present in aerosols and can be transmitted by means of aerosols. There is also a need for a detection device that can detect the presence of viruses in airborne aerosols and the viral content of the air for use in ambient air monitoring and alarming. The conventional PCR nucleic acid detection equipment is often realized on the basis of test tubes, disposable biochips and the like, and the test tubes or the disposable biochips need to be frequently replaced, so that continuous automatic operation of the equipment is difficult to realize, and thus, the environmental air is difficult to be continuously monitored for a long time.

In view of the above, it is expected that the nucleic acid detecting apparatus will be operated continuously, but to achieve this, it is often necessary to employ a reusable flow channel system. In the testing process, if a positive sample appears, the residue of the positive substance may be left in the flow channel system after the detection is completed, and how to prevent the residue of the positive substance in the flow channel system from affecting the detection result of the subsequent sample is a big problem to be solved currently.

Disclosure of Invention

The object of the present invention is to overcome the disadvantages of the prior art and to provide a solution for a continuously operating nucleic acid detection device that allows the removal of positive residues.

To solve the above technical problems, the present invention provides a nucleic acid detecting apparatus capable of continuously operating, comprising: the nucleic acid amplification reaction module comprises a micro-fluidic chip, and a sample mixed solution to be detected can be injected into a flow channel and an amplification reaction chamber of the micro-fluidic chip and carries out nucleic acid amplification reaction in the amplification reaction chamber; the fluorescence detection device is suitable for detecting a fluorescence signal of the amplification reaction chamber so as to obtain the amount of an amplification product in the amplification reaction chamber in real time according to the fluorescence signal; and a residue removing module which comprises a high-temperature fluid cleaning module and is used for injecting fluid with the temperature of more than 200 ℃ into the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber when the detection result of the nucleic acid detection equipment is positive so as to remove positive residues.

Wherein the nucleic acid detecting apparatus further comprises: the sampling module is suitable for receiving the exhaled breath, collecting the bioaerosol carried by the exhaled breath through the collection liquid and forming liquid-phase sample liquid; the sample sending module is used for pretreating and premixing the collected sample liquid and then conveying the mixed liquid to the nucleic acid amplification reaction module; wherein the pre-processing comprises: performing inactivation treatment and nucleic acid extraction treatment, wherein the premixing comprises the step of mixing a sample solution subjected to nucleic acid extraction with a reaction solution to obtain a sample mixed solution to be detected; when the detection result of the nucleic acid detection device is positive, the high-temperature fluid cleaning module is further used for injecting the fluid with the temperature of more than 200 ℃ into the flow channel and the container of the sample feeding module, which are used for pretreating and premixing the sample liquid, and further injecting the fluid with the temperature of more than 200 ℃ into the amplification reaction chamber of the nucleic acid amplification reaction module and the flow channel communicated with the amplification reaction chamber, so as to simultaneously remove positive residues in the sample feeding module and the nucleic acid amplification reaction module.

The working medium of the high-temperature fluid cleaning module is gas.

The working medium of the high-temperature fluid cleaning module is liquid.

The working medium of the high-temperature fluid cleaning module is silicone oil with the working temperature higher than 200 ℃ or engine oil with the working temperature higher than 200 ℃.

And the residue removing module is also used for injecting an extracting solution for nucleic acid extraction treatment into the sample sending module and the nucleic acid amplification reaction module when the detection result of the nucleic acid detection equipment is positive, dissolving or flushing positive residues in the flow channel and the amplification reaction chamber, and then removing the positive residues through the high-temperature fluid cleaning module.

The sample sending module comprises a plurality of containers for containing various types of liquid required by the pretreatment, a plurality of mixing pools required by the pre-mixing, hoses for connecting the containers and the mixing pools, and a plurality of peristaltic pumps for driving the liquid in the hoses to flow; the multiple containers comprise reaction liquid pools, positive liquid pools, negative liquid pools and extraction liquid pools, and the multiple mixing pools comprise nucleic acid extraction pools, positive control pools, negative control pools and sample pools to be detected; the residue removing module is used for injecting fluid with the temperature of more than 200 ℃ into the pipeline to be cleaned when the detection result of the nucleic acid detection equipment is positive, and the pipeline to be cleaned at least comprises: the nucleic acid extraction pool, the sample pool to be detected and a hose communicated with the nucleic acid extraction pool and the sample pool are arranged on the sample conveying module, and the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber are arranged on the sample conveying module.

The high-temperature fluid cleaning module is suitable for injecting fluid with the temperature of more than 200 ℃ into a pipeline to be cleaned in a high-pressure pulse mode; the nucleic acid detection equipment also comprises a gas injection module, wherein the gas injection module is communicated with the sample delivery module and is used for injecting gas into a flow channel of the sample delivery module through an injection port, and each injection port is provided with a valve; the valve is suitable for closing the flow channel of the sample sending module and opening the air channel of the gas injection module so as to allow gas to be injected into the flow channel of the sample sending module; or opening the flow channel of the sample sending module and closing the air channel of the gas injection module to allow the liquid in the flow channel of the sample sending module to pass through; the gas injection module and the high-temperature purging module share the same compressed gas cylinder, and the compressed gas cylinder is used for providing gas injected into the sample sending module.

Wherein, the sample sending module and the nucleic acid amplification reaction module are integrated in the microfluidic chip.

Wherein at least one part of the sections of the flow channels to be cleaned of the sample feeding module and the nucleic acid amplification reaction module are provided with heat insulation layers; the pipe to be cleaned at least comprises: the nucleic acid extraction pool of the sample sending module, the sample pool to be detected, a flow channel communicated with the sample pool, the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber.

Compared with the prior art, the application has at least one of the following technical effects:

1. the nucleic acid detection equipment can automatically remove the positive residues in the equipment after completing the detection of the positive samples, thereby avoiding the problem of false positive in the detection of subsequent samples.

2. The method can remove the positive substance residue in the runner system in a high-temperature gas purging mode, and the high-temperature gas purging is a physical removal method, so that the use cost can be obviously reduced compared with a chemical reagent residue removal mode.

3. The invention can crack the nucleic acid of virus (or other pathogens) by means of high-temperature gas purging,

the PCR amplification reaction capacity is no longer possessed, so that the residual positive substances cannot influence the detection result of the subsequent sample.

4. The invention can elute the liquid flow channel system by using extracting solution (such as deionized water) after each positive result appears, and then carry out high-temperature gas purging on the liquid flow channel, and the residue removing mode is favorable for reducing the requirement of equipment on the temperature of the high-temperature gas. Further, the above-mentioned elution and purging may be alternately performed and repeated a plurality of times (e.g., three times) to completely remove the positive residues.

5. The nucleic acid detection equipment provided by the invention realizes automatic operation in all links from sample collection to detection result output, and the usability of the nucleic acid detection equipment is obviously improved.

6. The invention obtains a real-time amplification curve based on nucleic acid isothermal amplification reaction and fluorescence detection, and has high accuracy and sensitivity.

7. The invention can continuously and continuously sample and continuously work, and can be used for detecting the nucleic acid of virus (or other pathogens) in aerosol in the air, thereby carrying out biological safety early warning on indoor closed space or occasions with dense personnel.

8. The invention can reduce the consumption of nucleic acid detection reagent, thereby reducing the use cost of equipment and reducing the workload and the working difficulty of treating biological waste liquid.

9. The nucleic acid detection device of the invention can be suitable for nucleic acid detection of new coronavirus, and also can be suitable for nucleic acid detection of influenza virus or other pathogens.

Drawings

FIG. 1 is a schematic block diagram of a nucleic acid detecting apparatus capable of continuous operation according to an embodiment of the present invention;

FIG. 2 shows a schematic diagram of a sample presentation module according to an embodiment of the invention;

FIG. 3 shows a schematic top view of a microfluidic chip in an embodiment of the invention;

FIG. 4 shows a schematic side cross-sectional view of a microfluidic chip in an embodiment of the invention;

fig. 5 shows a schematic side cross-sectional view of a microfluidic chip in another embodiment of the invention;

fig. 6 shows a schematic view of a microfluidic chip provided with a thermal insulation layer in another embodiment of the present invention.

Detailed Description

For a better understanding of the present application, various aspects of the present application will be described in more detail with reference to the accompanying drawings. It should be understood that the detailed description is merely illustrative of exemplary embodiments of the present application and does not limit the scope of the present application in any way. Like reference numerals refer to like elements throughout the specification. The expression "and/or" includes any and all combinations of one or more of the associated listed items.

It should be noted that the expressions first, second, etc. in this specification are used only to distinguish one feature from another feature, and do not indicate any limitation on the features. Thus, a first body discussed below may also be referred to as a second body without departing from the teachings of the present application.

In the drawings, the thickness, size, and shape of an object have been slightly exaggerated for convenience of explanation. The figures are purely diagrammatic and not drawn to scale.

It will be further understood that the terms "comprises," "comprising," "includes," "including," "has," "including," and/or "including," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Moreover, when a statement such as "at least one of" appears after a list of listed features, the entirety of the listed features is modified rather than modifying individual elements in the list. Furthermore, when describing embodiments of the present application, the use of "may" mean "one or more embodiments of the present application. Also, the term "exemplary" is intended to refer to an example or illustration.

As used herein, the terms "substantially," "about," and the like are used as terms of table approximation and not as terms of table degree, and are intended to account for inherent deviations in measured or calculated values that will be recognized by those of ordinary skill in the art.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

The invention is further described below with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic block diagram of a nucleic acid detecting apparatus capable of continuous operation according to an embodiment of the present invention. Referring to fig. 1, according to an embodiment of the present invention, there is provided a nucleic acid detecting apparatus capable of continuous operation, which includes a sampling module, a sample sending module, a nucleic acid amplification reaction module, a lock phase detecting device, and a residue removing module. Wherein the sampling module may be an exhaled breath collection device, such as an exhaled breath condensate collector. The exhaled breath condensate collector may include a bellows (or other gas conduit) that may have an exhalation nozzle to facilitate exhalation by the patient (or subject), as well as an exhalation mask. With the help of the air pump, the exhaled air enters the collection tube through the bellows. The collection tube may have a Virus storage buffer (Virus storage buffer) in it. The virus preservation buffer can collect virus particles in the aerosol into a liquid, thereby forming a virus-containing sample in a liquid phase (hereinafter sometimes referred to as a sample liquid). And the collection process can avoid the damage or pollution of the nucleic acid of the virus particles, thereby ensuring the accuracy of the detection result. It should be noted that the sampling module of the present invention is not limited to the exhaled breath condensate collector, as long as the sampling module can receive exhaled breath and collect the biological aerosol carried by the exhaled breath through a collection liquid (e.g., a virus preservation buffer) to form a liquid phase virus-containing sample. Further, in this embodiment, the sample sending module is used for pre-treating and pre-mixing the sample solution collected by the sampling module, and then delivering the mixed solution to the nucleic acid amplification reaction module. Wherein, the pre-treating the sample liquid may include: inactivation treatment and nucleic acid extraction treatment, wherein the pre-mixing comprises mixing the sample solution after the nucleic acid extraction with the reaction solution. Further, the sample sending module can be used for providing and sending positive control mixed liquor and negative control mixed liquor for the nucleic acid amplification reaction module. In a specific implementation, the sample sending module can comprise a plurality of containers for containing various types of liquid, a plurality of mixing pools, hoses for connecting the containers and the mixing pools, and a plurality of peristaltic pumps for driving the liquid in the hoses to flow. Further, in this embodiment, the nucleic acid amplification reaction module may be implemented based on a microfluidic biochip (hereinafter sometimes referred to as a microfluidic chip). The sample mixture (i.e. the mixture obtained by pre-treating and pre-mixing the sample solution) to be detected provided by the sample sending module is injected into the microfluidic biochip through a hose, and a nucleic acid amplification reaction is performed in a reaction chamber (which may be referred to as an amplification reaction chamber) of the microfluidic biochip. The phase-locked detection device is used for detecting the fluorescence signal of the amplification reaction chamber, and then obtaining the amount of the amplification product in the amplification reaction chamber in real time according to the fluorescence signal. Further, in order to remove the positive residues and realize the sustainable work of the equipment, the nucleic acid detection equipment of the embodiment further comprises a residue removing module, wherein the residue removing module can comprise a high-temperature purging module, and the high-temperature purging module comprises an air pump, a compressed air cylinder and a corresponding control module. The compressed gas cylinder can be communicated with the flow channel system of the nucleic acid amplification reaction module through a valve. When the nucleic acid detection equipment is in a working state of sample detection, the valve is closed, and gas in the compressed gas cylinder cannot enter the flow channel system. The nucleic acid detection equipment can normally collect a sample, extract nucleic acid and mix reaction liquid to obtain a sample liquid to be detected, and the sample liquid to be detected is injected into an amplification reaction chamber of the microfluidic chip to carry out constant-temperature amplification reaction. When the detection result is negative, the next sample collection and detection can be continued. And when the detection result is positive, starting the high-temperature purging module to remove the positive residues. Specifically, the residual liquid in the flow channel system and the amplification product of the positive sample solution in the chip are normally discharged first, i.e., the normal waste liquid discharge operation for each detection is completed. Then, a valve of the high-temperature purging module is opened, high-temperature gas is blown into the flow channel system by the air pump and then purged along the flow channel formed by the hose, in the purging process, the high-temperature gas can be injected into each mixing tank to perform high-temperature degradation on residual positive substances in the mixing tanks, and the high-temperature gas can also be injected into the microfluidic chip through the hose, so that the flow channel and the amplification reaction chamber in the microfluidic chip are purged. The purge time may last 30 minutes or more. The temperature of the high-temperature gas may be a preset temperature capable of degrading DNA. For DNA nucleic acids, the degradation temperature is about 200 ℃. In the case of RNA nucleic acids (e.g., RNA viral nucleic acids), since RNA viral nucleic acids are amplified based on an RT-PCR reaction system, that is, the RNA nucleic acids are reverse-transcribed into cDNA templates and then cyclically amplified, and the positive residues in the amplification reaction chamber contain a large amount of amplification products after reverse transcription, the degradation temperature of high-temperature degradation is also set to about 200 ℃ when RNA nucleic acids (e.g., new coronavirus nucleic acids) are detected, so as to degrade cDNA amplification products. In this embodiment, the high temperature purge module may be equipped with a heating unit to provide a high temperature gas having a set temperature.

Further, fig. 2 shows a schematic diagram of a sample sending module according to an embodiment of the present invention. Referring to fig. 2, in the present embodiment, the sample feeding module includes a nucleic acid extraction pool, a reaction liquid pool, a positive liquid pool, a negative liquid pool, a positive control pool, a negative control pool, a sample mixing pool (also sometimes referred to as an amplification reaction pool) to be tested, a plurality of hoses for conveying liquid between different containers (pools), and a plurality of peristaltic pumps for driving the liquid flow in the hoses. The sampling module can be provided with a sample liquid pool (or other types of sample liquid containers), the sample liquid pool is communicated with the nucleic acid extraction pool through a hose, the nucleic acid extraction pool can be provided with or injected with nucleic acid extracting solution, and the nucleic acid extracting solution and the sample liquid are fully mixed in the nucleic acid extraction pool. In this embodiment, the nucleic acid extracting solution may be deionized water or other specific extracting solution corresponding to the target virus. The sample solution is mixed with the nucleic acid extract solution, and the nucleic acid is extracted from the viral particles by a high-temperature treatment for a predetermined period of time (for example, a high-temperature treatment at about 95 ℃ for 10 minutes for the new coronavirus), thereby obtaining a sample solution suitable for nucleic acid release for an amplification reaction. The amplification reaction tank can be respectively communicated with the nucleic acid extraction tank and the reaction liquid tank, so that the sample liquid released by the nucleic acid and the reaction liquid are mixed in the amplification reaction tank to obtain a sample to be detected for carrying out amplification reaction. The positive control pool can be respectively communicated with the reaction liquid pool and the positive liquid pool, so that the positive control liquid is obtained in the positive control pool. The negative control pool can be respectively communicated with the negative liquid pool and the reaction liquid pool, so that the negative control liquid is obtained in the negative control pool. It should be noted that the selection of the positive control solution and the negative control solution is not unique. The communication between the different containers can be realized by hoses, and the flowing direction and the flowing quantity of the liquid in each hose are controlled by a peristaltic pump.

It should be noted that in the above-mentioned examples, although deionized water was used as the nucleic acid extracting solution, the present invention is not limited thereto. For example, in another embodiment of the present invention, the nucleic acid extracting solution may comprise a lysis solution, and after the lysis solution is mixed with the sample solution, the structures such as the envelope and/or capsid of the virus particle can be lysed, so as to release the nucleic acid and simultaneously perform the virus inactivation function. After the sample solution is mixed with the lysate, the sample solution suitable for the release of the nucleic acid for the amplification reaction can be obtained through the steps of extraction, elution (e.g., the lysate is eluted), and the like.

Further, in one embodiment, the sample module may further comprise an extraction liquid reservoir, which may be used to contain a nucleic acid extraction liquid. The extraction solution reservoir may be in communication with the nucleic acid extraction reservoir for delivering nucleic acid extraction solution to the nucleic acid extraction reservoir. In another embodiment, when the nucleic acid extraction includes multiple steps of lysis, extraction, elution, etc., the sample feeding module may further include a lysis solution pool, an extraction solution pool, an elution solution pool, etc., and the lysis solution pool, the extraction solution pool, and the elution solution pool may be respectively communicated with the nucleic acid extraction pool.

In the above embodiment, the reaction solution may include a primer corresponding to the target nucleic acid and an enzyme (for example, reverse transcriptase, polymerase, and the like may be included). The positive solution may contain the target nucleic acid at a known concentration, and the positive control solution may provide a real-time amplification curve for the positive control channel in the nucleic acid detection device. The positive control channel may be disposed in a microfluidic chip. The negative control mixture can be injected into a negative reaction chamber in the microfluidic chip to provide a real-time amplification curve of the negative control channel. The real-time amplification curves of the positive control channel and the negative control channel are measured, so that the virus concentration of the sample can be quantitatively measured more accurately or the nucleic acid detection equipment can be corrected. In another embodiment, the negative liquid (i.e., not mixed with the reaction liquid) can also be directly injected into the negative reaction chamber of the microfluidic chip to provide a real-time amplification curve of the negative control channel.

The nucleic acid detection equipment of the embodiment can realize complete self-service nucleic acid detection, namely, the automatic operation is realized in all links from sample collection to detection result output, so that the usability of the nucleic acid detection equipment is obviously improved. The detection result can be displayed on a display screen configured by the nucleic acid detection equipment, and also can be transmitted to a machine room and interpreted by professionals in the machine room. Since the interpretation personnel can be kept away from the nucleic acid detection device and the patient, or kept isolated from the patient at all times, the risk of infection is reduced.

Further, in an embodiment of the present invention, the integrated machine may further include a heating module for providing a temperature required for nucleic acid extraction and isothermal amplification reaction. Wherein, the temperature required for nucleic acid extraction may be 95 ℃. The temperature required for isothermal amplification reaction may be 60 ℃. Still further, the all-in-one machine can further comprise a refrigeration module, and the refrigeration module is used for providing the temperature suitable for preserving the extracting solution, the reaction solution and the positive solution. In one embodiment, the temperature suitable for preserving the nucleic acid extracting solution, the reaction solution and the positive solution may be 0 to 4 ℃.

Further, fig. 3 shows a schematic top view of a microfluidic chip according to an embodiment of the present invention. Referring to fig. 3, in this embodiment, the nucleic acid amplification reaction module includes a microfluidic chip 1, and the microfluidic chip 1 includes three independent flow channels, that is, a detection flow channel 2a of a sample to be detected, a positive control flow channel 2b, and a negative control flow channel 2 c. The positive control flow path 2b and the negative control flow path 2c each have one reaction chamber 3. The sample-detecting flow path 2a to be measured may have a plurality of reaction chambers 3 connected in series. Further, the sample detection flow channel 2a may have a serpentine shape, the serpentine shape is a combination of a plurality of "S" shapes, and a plurality of reaction chambers 3 (which may be referred to as amplification reaction chambers) for amplification reaction may be connected in series by the serpentine sample detection flow channel, wherein each turn section of the sample detection flow channel 2a may be provided with one amplification reaction chamber. The microfluidic chip 1 may also have threaded positioning holes 8 to facilitate fixing the microfluidic chip 1 in place inside the integrated machine. The screw positioning hole 8 may be provided in plural, and the position thereof may be set so as to avoid the flow channels and the reaction chamber. In this embodiment, in order to better perform pre-mixing or pumping, the volume of the liquid received in the sample cell to be detected each time is generally significantly over 4 microliters, and the arrangement of the serpentine flow channel and the plurality of amplification reaction chambers connected in series is helpful to effectively utilize the precious reagent to improve the detection accuracy. For example, the integrated machine may be configured such that the detection result is determined to be negative only when all amplification reaction chambers of the serpentine flow channel are negative. If any reaction chamber is positive, the detection result is judged to be positive. Further, in another embodiment, a plurality of amplification reaction chambers with serpentine flow channels can be used to improve the accuracy of viral quantitative nucleic acid detection. For example, the Ct values of a plurality of positive reaction chambers can be averaged as an output. According to the output Ct value and the configuration of the all-in-one machine, the virus concentration contained in the collected sample can be calculated. On the other hand, the liquid injected into each reaction chamber may be affected by the factors such as gravity, residual air and non-uniform dynamic flow resistance, and the amplification reaction chambers of the detection flow channel of the sample to be detected are arranged in series in the embodiment, which is helpful to reduce the interference of the factors on the detection result. For example, if the amplification reaction chambers are arranged in parallel, gravity, residual air, uneven dynamic flow resistance, etc., may cause some amplification reaction chambers to be filled with no liquid or to have too little liquid, and other amplification reaction chambers to be filled with too much liquid, which may lead to a deviation in the detection result. And a series design may avoid or reduce such deviations. In addition, in this embodiment, the positive control flow channel, the negative control flow channel and the to-be-detected sample detection flow channel have independent liquid inlets 6 and independent liquid outlets 7, that is, the three flow channels are respectively led out of the microfluidic chip through three hoses. The design can avoid cross contamination caused by public effluent.

Further, fig. 4 shows a schematic side cross-sectional view of a microfluidic chip in an embodiment of the present invention. In this embodiment, the microfluidic chip includes a substrate 5 and a cover plate 4, wherein the reaction chamber 3 and the flow channel 2 can be processed on the upper surface of the substrate 5 (the flow channel 2 includes the aforementioned detection flow channel 2a, positive control flow channel 2b, and negative control flow channel 2c for the sample to be detected), the lower surface of the cover plate 4 can be thermally bonded to the upper surface of the substrate 5, and the joint between the side surfaces of the substrate 5 and the cover plate 4 can be glued to seal the side surfaces. The cover plate 4 may have a plurality of through holes, which may serve as a liquid inlet 6 and a liquid outlet 7 for each flow channel (see fig. 3). Note that the through-hole of the cover plate 4 is not in the cross-section shown in fig. 4, so it is not shown in fig. 4.

Fig. 5 shows a schematic side cross-sectional view of a microfluidic chip in another embodiment of the present invention. Referring to fig. 5, the liquid inlet 6 is a through hole provided in the cover plate 4, and the through hole communicates with the flow channel 2. The arrangement mode of the liquid outlet can be consistent with that of the liquid inlet, and the detailed description is omitted.

Further, still referring to fig. 2, in one embodiment of the present invention, the nucleic acid amplification reaction apparatus may further include a heating module for providing a temperature required for nucleic acid extraction and isothermal amplification reaction. Wherein, the temperature required for nucleic acid extraction may be 95 ℃. The temperature required for isothermal amplification reaction may be 60 ℃. Still further, the nucleic acid amplification reaction apparatus may further include a refrigeration module for providing a temperature suitable for preserving the extraction solution, the reaction solution, and the positive solution. In one embodiment, the temperature suitable for preserving the extract, the reaction solution and the positive solution may be 0 to 4 ℃.

Further, in one embodiment of the present invention, the extracting solution used by the nucleic acid detecting apparatus may be deionized water. In the residue removing step, deionized water can be used for dissolving or flushing the residual positive substances to a certain degree, so that the high-temperature purging module is assisted to thoroughly remove the nucleic acids of the residual positive substances. It should be noted that in normal sample detection process, the extract usually has a certain cleavage function, but the cleavage in the extraction step mainly means the cleavage of the envelope, capsid or other structural protein of the virus particle to help release the virus nucleic acid to be detected. Whereas what the high temperature purge step needs to degrade is the nucleic acid itself. After the nucleic acid is degraded, the capability of amplification reaction is no longer available, so that the influence of positive residue on the next detection result can be avoided. In this embodiment, when the detection result is negative, the next sample collection and detection can be continued. When the detection result is positive, the flow channel system is eluted by deionized water, and then is purged by high-temperature gas, so that the nucleic acid of the residual positive substances is thoroughly degraded. The purge time may last 30 minutes or more. The temperature of the high-temperature gas may be a preset temperature capable of degrading the DNA or cDNA amplification product. Generally, the temperature of the high-temperature gas is 200 ℃ or higher. The high temperature purge module may be equipped with a heating unit to provide a high temperature gas having a set temperature. In the actual test using the pseudovirus with higher concentration, the hose and the microfluidic chip are eluted by deionized water, and then are treated for 30 minutes by using high temperature, and no positive residual substance is detected in the hose or the microfluidic chip.

Further, in an embodiment of the present invention, the operation mode of the residue removing module may be: and when the detection result is positive, eluting the flow channel system by using deionized water, then purging by using high-temperature gas, eluting by using deionized water, then purging by using high-temperature gas, and alternating the steps so as to finally thoroughly crack the nucleic acid of the residual positive substances. The total length of the purge time may last 30 minutes or more. The temperature of the high-temperature gas may be a preset temperature capable of degrading the DNA or cDNA amplification product. Generally, the temperature of the high-temperature gas is 200 ℃ or higher. The high temperature purge module may be equipped with a heating unit to provide a high temperature gas having a set temperature.

Further, in one embodiment of the present invention, the heating unit of the high temperature purge module may heat the gas to more than 200 ℃. Because the high-temperature gas can be cooled to a certain degree in the process of passing through the runner system, the temperature setting higher than 200 ℃ can prevent the degradation temperature required by insufficient gas temperature received by a hose and a micro-fluidic chip which are positioned at the rear end of the runner system.

Further, fig. 6 shows a schematic view of a microfluidic chip provided with an insulating layer in another embodiment of the present invention. Referring to fig. 6, the microfluidic chip may have an insulating layer 7, and the insulating layer 7 may wrap the cover plate 4 and the substrate 5 of the microfluidic chip 1. The heat-insulating layer 7 is arranged on the microfluidic chip 1, so that heat leakage of a pipeline system can be reduced, and the problem that nucleic acid at the tail end of a pipeline cannot be degraded due to overlarge temperature drop of high-temperature gas of the high-temperature purging module is avoided. In this embodiment, the insulating layer may be made of an insulating material, or may be a vacuum interlayer. It should be noted that, at the positions of the amplification reaction chambers corresponding to the microfluidic chip, and the reaction chambers of the negative control flow channel and the positive control flow channel, the insulating layer is provided with an avoiding structure or is set to be in a transparent state, so that each reaction chamber receives the irradiation of the excitation source module and detects the fluorescence signal of each reaction chamber. Furthermore, each hose of the sample sending module can also be wrapped with an insulating layer, and the insulating layer can be made of insulating materials or can be a vacuum interlayer. In this embodiment, add the heat preservation and help reducing the temperature drop of high temperature gas (referring to the high temperature gas that high temperature sweeps the module and provide) from the air inlet to the gas outlet to prevent that the difference in temperature between air inlet department and gas outlet department is too big. If the temperature drop of the high-temperature gas from the gas inlet to the gas outlet is too large, the gas temperature at the gas inlet is inevitably required to be raised in order to meet the requirement of nucleic acid degradation at the tail end of the flow channel, so that the requirement of a sample sending module (such as a hose) has better heat resistance, the material selection is difficult, and the energy conservation is not facilitated. And after the heat-insulating layer is arranged, the problems can be effectively solved or alleviated.

Further, in one embodiment of the present invention, the flexible tube and the microfluidic chip of the flow channel system of the nucleic acid detecting apparatus are both made of a heat-resistant material. For example, microfluidic chips can be fabricated using glass or PC. Generally, glass can provide better heat resistance and reliability.

Still further, in an embodiment of the present invention, the residue removing module may further include a polishing solution eluting module, and the polishing solution eluting module may include a polishing solution storage tank and a hose and a peristaltic pump for connecting the polishing solution storage tank to the sample feeding module or injecting the polishing solution into the microfluidic chip. The polishing solution elution module can be used when positive residues still exist after the high-temperature purging module is purged. For example, the nucleic acid extract may be eluted, and then purged at a high temperature, and then whether the flow channel still has a positive residue is detected, and if so, the polishing solution elution module is started, and the polishing solution is injected to elute the flow channel and the reaction chamber. The polishing liquid may be a commercially available polishing liquid containing fine particles, or may be self-prepared, for example, in one example, a synthetic diamond powder may be mixed with water and an auxiliary agent to prepare the polishing liquid.

In another embodiment of the present application, the residue removing module may include a high-temperature liquid injection module, and the high-temperature liquid injection module may replace the high-temperature purging module using gas as the working medium in the foregoing embodiments. Compared with a gas working medium, the liquid working medium generally has higher specific heat, which is beneficial to reducing the heat loss of the working medium in the process of cleaning a flow passage, namely reducing the temperature difference from the inlet end to the outlet end of the working medium. The liquid working medium may be, for example, an oily liquid having an operating temperature of 200 ℃ or higher. For example, in one example, the working fluid of the high temperature liquid injection module may be Silicone Oil (Silicone Oil) having a formula of C₁₆H₂₂O₂Si₂The working temperature of the silicone oil can reach 250 ℃. In practice, the temperature of the silicone oil at the inlet end may be below 250 ℃. The temperature of the silicone oil at the inlet end can be set, for example, according to the actual conditions in terms of length of the pipe to be cleaned (i.e. to remove residues), flow resistance, heat losses, etc., as long as it is ensured that the temperature of the silicone oil at the outlet section is higher than 200 ℃. Currently, silicone oil on the market is sold at a price of about hundreds of dollars per gram, and the price of nucleic acid washing enzyme is usually above hundreds of dollars per milligram, so that the scheme of the embodiment has a significant cost advantage (the cost of the two is an order of magnitude difference) compared with the existing residue removing scheme of nucleic acid washing enzyme and the like, and the use cost of equipment can be greatly reduced. Furthermore, the scheme of the embodiment can also omit the subsequent cleaning work of the nucleic acid cleaning enzyme, and can avoid the false negative problem caused by the residual nucleic acid cleaning enzyme.

Further, in another embodiment of the present invention, engine oil may be used to replace the silicone oil as the liquid working medium of the high temperature liquid injection module. For example, the engine oil used in the engine has the working temperature of more than 400 ℃ and can meet the temperature requirement of high-temperature cleaning (namely more than 200 ℃). And the engine oil is low in cost, and has a remarkable cost advantage compared with the existing residue removal schemes such as nucleic acid cleaning enzyme. Meanwhile, the embodiment can also omit the subsequent cleaning work of the nucleic acid cleaning enzyme, and can avoid the false negative problem caused by the residual nucleic acid cleaning enzyme.

For ease of description, the high temperature purge module and the high temperature liquid injection module herein may be collectively referred to as a high temperature fluid purge module.

Further, in another embodiment of the present invention, the residue removing module may still adopt a high temperature purging module, and the working medium thereof is still a gas working medium. The difference from the previous embodiment is that the present embodiment uses high-pressure pulse to inject gas into the pipe to be cleaned. When gas is injected, the high-pressure pulse can generate gas explosion, so that the flow resistance problem and the heat loss problem of the gas passing through a pipeline to be cleaned are overcome. On the other hand, since the flow passage of the apparatus is very thin in this embodiment and the total volume of the flow passage to be cleaned is small, the amount of gas and pressure required for the gas explosion do not need to be large, and the noise thereof can be controlled within a reasonable range, for example, the noise thereof can be on the same level as that of the mechanical pump. Further, in one embodiment of the present invention, a sound absorbing material may be disposed around the sample sending module and the microfluidic chip to further reduce noise generated by the air explosion. The sound absorbing material may be a sound dampening foam.

In the nucleic acid amplification reaction apparatus according to the above embodiment, the microfluidic chips each have a detection channel (the detection channel includes an amplification reaction chamber and an input/output flow channel connecting the amplification reaction chamber), a negative control channel, and a positive control channel. It should be noted that the microfluidic chip of the present invention is not limited thereto. For example, in some embodiments of the invention, a microfluidic chip may have only detection channels, or only detection channels and negative (or positive) control channels. In these embodiments, the negative control real-time amplification curve and/or the positive control real-time amplification curve can be pre-measured and calibrated and then stored in the nucleic acid detection device. During the amplification reaction of the sample to be detected, the real-time amplification curve of the sample to be detected can be obtained from the fluorescence signal detected by the detection channel, and the real-time amplification curve is compared with the stored and pre-calibrated negative control real-time amplification curve and/or positive control real-time amplification curve, so that a positive or negative detection result or a quantitative detection result of the virus concentration can be obtained.

Further, in one embodiment of the present invention, the amount of the amplification product in the amplification reaction chamber is detected based on the detection of the fluorescence signal in the amplification reaction chamber by the phase lock detection device. The photoelectric probe of the phase-locking detection device can be arranged at a position close to the amplification reaction chamber, and an optical filter can be arranged between the amplification reaction chamber and the photoelectric probe to filter the interference of excitation laser for exciting fluorescence. In other embodiments of the present invention, the lock detection device can also be implemented by a fluorescence detection device commonly used in a PCR detector, as long as the fluorescence signal in the amplification reaction chamber can be detected.

Further, in an embodiment of the present invention, the nucleic acid detecting apparatus may further include a gas injection module, which is in communication with the sample sending module, for injecting a gas into the flexible tube of the sample sending module. The injection port may be provided between the reagent reservoir and the mixing reservoir, for example, between the extraction liquid reservoir and the nucleic acid extraction reservoir, or between the reaction liquid reservoir and the sample reservoir to be measured. A two-way valve can be arranged at each injection port, and the two-way valve can close the flow channel of the sample sending module and open the air channel of the gas injection module, so that gas can be injected into the flexible pipe of the sample sending module; or opening the sample sending module flow passage and closing the air passage of the gas injection module, so that the liquid in the hose of the sample sending module can pass through. This embodiment can effectively reduce the reagent consumption per nucleic acid detecting process. Specifically, in the present invention, the microfluidic chip is used for nucleic acid amplification reaction and nucleic acid detection, and the amount of liquid required in the reaction chamber can be as small as several microliters in each nucleic acid detection. However, since the flexible tube of the sample feeding module has a certain volume, a large amount of various precious reagents such as reaction solution and extraction solution may remain in the flexible tube of the sample feeding module after the nucleic acid detection is completed, which causes a problem of high reagent consumption. In this embodiment, the output of the reagent can be controlled by controlling the two-way valve, for example, only a few microliters of reaction solution can be provided at each detection, then the sample sending module flow channel is closed to prevent the reaction solution from being output, and meanwhile, the gas channel of the gas injection module is opened to inject gas into the sample sending module. When the next detection is carried out, the flow channel is opened, the air channel is closed, and microliter reaction liquid required by a single reaction is output. Then the flow passage is closed again, and the air passage is opened. The above cycle is performed to precisely control the output of the reaction solution in a single reaction. With the solution of this embodiment, the liquid in the hose may be in the form of a plurality of liquid segments, with adjacent liquid segments being separated by gas. Further, in one embodiment, the gas injection module may include a compressed gas cylinder for providing gas for injection into the sample presentation module. Still further, the gas injection module and the high temperature purge module may share the same compressed gas cylinder. In the normal detection mode, the compressed gas cylinder provides normal-temperature gas, and in the residue removing mode, the compressed gas cylinder provides high-temperature gas.

Further, in some embodiments of the present invention, the sample feeding module and the nucleic acid amplification reaction module may be integrated into a microfluidic chip. For example, the sample sending module and the nucleic acid amplification reaction module can be integrated in the same microfluidic chip, the sampling module can be realized by a disposable device, and each patient or subject can finish sample collection by the disposable sampling module, so that the problem of cross infection can be avoided, and the safety of equipment can be enhanced. The sample sending module and the nucleic acid amplification reaction module are integrated in the same microfluidic chip, so that the injection of high-temperature fluid can be facilitated to realize the function of removing positive residues. When the sample sending module and the nucleic acid amplification reaction module can be integrated in the same microfluidic chip, the use of an additional hose made of a high-temperature-resistant material can be avoided, the high-temperature-resistant capacity of the microfluidic chip is only considered, and generally, the microfluidic chip made of a glass material can resist the high temperature of more than 200 ℃, so that the sample sending module and the nucleic acid amplification reaction module have good safety and reliability. For another example, the sample sending module and the nucleic acid amplification reaction module may be integrated into different microfluidic chips, respectively, and the different microfluidic chips may be connected using a hose or other type of conduit. Although a hose or other types of connection pipes still need to be used, compared with a sampling module using a large number of hoses, the sampling module integrated on the microfluidic chip of the present embodiment can significantly reduce the length and number of connection hoses that need to be made of high temperature resistant materials, thereby facilitating to reduce the difficulty of implementing the residue removing module of the device and improving the safety and reliability of the residue removing module. In summary, the solution of integrating the sample sending module and the nucleic acid amplification reaction module into the microfluidic chip can be regarded as including the following two cases: in a first scheme, the sample sending module and the nucleic acid amplification reaction module can be integrated in the same microfluidic chip; and in the second scheme, the sample sending module and the nucleic acid amplification reaction module are respectively integrated in different microfluidic chips, and the different microfluidic chips are connected by using hoses or other types of pipelines.

Further, in one embodiment of the present invention, an insulating layer may be provided on at least a portion of the flow channel to be cleaned of the sample feeding module and the nucleic acid amplification reaction module. The pipe to be cleaned at least comprises: the nucleic acid extraction pool of the sample sending module, the sample pool to be detected, a flow channel communicated with the sample pool, the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber. In this embodiment, the insulating layer may be disposed in all sections of the flow channel to be cleaned, or may be disposed in only a part of sections of the flow channel to be cleaned, so long as it is ensured that the temperature drop of the working medium injected by the high-temperature fluid cleaning module in the pipe to be cleaned meets a preset index, so that the temperature of the working medium at the outlet of the flow channel to be cleaned is still maintained above 200 ℃.

Further, in one embodiment of the present invention, the amplification reaction of the amplification reaction chamber is an RT-PRC amplification reaction, i.e., a reverse transcription-polymerase chain amplification reaction. In this example, after extracting a nucleic acid fragment from a sample, a cDNA is first synthesized based on RNA of the extracted nucleic acid fragment by the action of a reverse transcriptase (i.e., reverse transcription is performed), and then a long cDNA chain (which may be referred to as an amplification product) is amplified and synthesized by a primer and a DNA polymerase in a PCR reaction system by using the cDNA as a template (which may be referred to as a cDNA template or a template cDNA). The amplification product contains a large number of cDNA templates (i.e., cDNA templates reverse transcribed from the original nucleic acid fragments) arranged in a circular array. In the amplification reaction process, the amplification product in the amplification reaction chamber is irradiated by laser, so that a large number of fluorescent probes (or other types of fluorescent labels) carried by cDNA templates can emit flickering fluorescence. In this embodiment, a fluorescence threshold may be set first. For example, the fluorescence signal of the first 15 cycles of the amplification reaction can be used as the fluorescence background signal, with the default setting of the fluorescence threshold being 10 times the standard deviation of the fluorescence signal for 3-15 cycles. When the fluorescence signal of the amplification product reaches the fluorescence threshold, the Ct value can be obtained through the detected real-time amplification curve, and further the concentration information of the prokaryotic acid fragment can be obtained through the Ct value. The Ct value may be translated as a cycle threshold. For each sample, the Ct value for that sample is linear with respect to the logarithm of the number of initial nucleic acid fragments it contains (alternatively referred to as the starting copy number). The higher the initial copy number, the lower the Ct value. A standard curve can be obtained using a standard with a known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Therefore, once the Ct value of an unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve. In this embodiment, the target nucleic acid fragment is a specific nucleic acid fragment of the new coronavirus, so the concentration information of the nucleic acid fragment can reflect the load of the new coronavirus in the sample.

Further, in another embodiment of the present invention, the amplification reaction in the amplification reaction chamber may be a LAMP reaction. LAMP is known as Loop-mediated isothermal amplification and can be translated as Loop-mediated isothermal amplification. When the target nucleic acid is an RNA viral nucleic acid (e.g., a novel coronavirus nucleic acid), the sample needs to be subjected to reverse transcription so as to become a cDNA template that can be subjected to LAMP reaction. Compared with a PCR reaction system, the LAMP reaction system can have a faster amplification speed. In the LAMP reaction system, 4 specific primers can be designed aiming at 6 regions of a target gene (in the embodiment, the target gene can be target virus nucleic acid), under the action of strand displacement DNA polymerase (Bst DNA polymerase), the constant temperature amplification is carried out at 60-65 ℃, the high-fold nucleic acid amplification can be realized within 15-60 minutes, and the LAMP reaction system has the characteristics of simple operation, strong specificity, easy detection of products and the like. In this embodiment, the phase-locked detection device based on source-probe separation can perform high-precision measurement on the fluorescent signal of the LAMP amplification product in the reaction chamber, so as to obtain a corresponding real-time amplification curve, and further obtain a qualitative or quantitative detection result of the sample to be detected. Furthermore, the nucleic acid detection speed of the sample can be further increased based on the previously described method for predicting the Ct value or predicting the qualitative or quantitative detection result.

Further, in yet another embodiment of the present invention, the amplification reaction in the amplification reaction chamber may be a CAMP reaction. CAMP is collectively referred to as Competitive and systematic isothermal amplification and can be translated into isothermal amplification based on Competitive complementary pairing. When the target nucleic acid is an RNA viral nucleic acid (e.g., a novel coronavirus nucleic acid), the sample needs to be subjected to reverse transcription so as to become a cDNA template that can be subjected to LAMP reaction. The CAMP reaction system may have a faster amplification rate relative to the PCR reaction system. Compared with an LAMP reaction system, the CAMP reaction system has the advantages of low primer design difficulty, low requirement on target nucleic acid and the like. In this embodiment, the phase-locked detection device based on source-probe separation can perform high-precision measurement on the fluorescence signal of the CAMP amplification product in the reaction chamber, so as to obtain a corresponding real-time amplification curve, and further obtain a qualitative or quantitative detection result of the sample to be detected. Furthermore, the nucleic acid detection speed of the sample can be further increased based on the previously described method for predicting the Ct value or predicting the qualitative or quantitative detection result.

The nucleic acid detection device of the invention can be suitable for nucleic acid detection of new coronavirus, and also can be suitable for nucleic acid detection of influenza virus or other pathogens. When detecting RNA virus, target nucleic acid is firstly reversely transcribed into cDNA and then isothermal amplification is carried out. In the case of detecting DNA viruses, the reverse transcription step is omitted.

Further, the nucleic acid detecting apparatus according to some embodiments of the present invention may also be used for detecting genomes, DNA or RNA of various other organisms for nucleic acid detection.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A nucleic acid detecting apparatus capable of continuously operating, comprising:

the nucleic acid amplification reaction module comprises a micro-fluidic chip, and a sample mixed solution to be detected can be injected into a flow channel and an amplification reaction chamber of the micro-fluidic chip and carries out nucleic acid amplification reaction in the amplification reaction chamber;

the fluorescence detection device is suitable for detecting a fluorescence signal of the amplification reaction chamber so as to obtain the amount of an amplification product in the amplification reaction chamber in real time according to the fluorescence signal; and

and the residue removing module comprises a high-temperature fluid cleaning module, and the high-temperature fluid cleaning module is used for injecting fluid with the temperature of more than 200 ℃ into the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber when the detection result of the nucleic acid detection equipment is positive so as to remove positive residues.

2. The nucleic acid detecting apparatus according to claim 1, characterized in that the nucleic acid detecting apparatus further comprises:

the sampling module is suitable for receiving the exhaled breath, collecting the bioaerosol carried by the exhaled breath through the collection liquid and forming liquid-phase sample liquid; and

a sample sending module for pretreating and premixing the collected sample liquid and then delivering the mixed liquid to the nucleic acid amplification reaction module; wherein the pre-processing comprises: performing inactivation treatment and nucleic acid extraction treatment, wherein the premixing comprises the step of mixing a sample solution subjected to nucleic acid extraction with a reaction solution to obtain a sample mixed solution to be detected;

when the detection result of the nucleic acid detection device is positive, the high-temperature fluid cleaning module is further used for injecting the fluid with the temperature of more than 200 ℃ into the flow channel and the container of the sample feeding module, which are used for pretreating and premixing the sample liquid, and further injecting the fluid with the temperature of more than 200 ℃ into the amplification reaction chamber of the nucleic acid amplification reaction module and the flow channel communicated with the amplification reaction chamber, so as to simultaneously remove positive residues in the sample feeding module and the nucleic acid amplification reaction module.

3. The nucleic acid detecting apparatus according to claim 2, wherein the working medium of the high-temperature fluid cleaning module is a gas.

4. The nucleic acid detecting apparatus according to claim 2, wherein the working medium of the high-temperature fluid cleaning module is a liquid.

5. The nucleic acid detection device of claim 4, wherein the working medium of the high-temperature fluid cleaning module is silicone oil with a working temperature higher than 200 ℃ or engine oil with a working temperature higher than 200 ℃.

6. The nucleic acid detecting apparatus according to claim 2, wherein the residue removing module is further configured to inject an extracting solution for a nucleic acid extraction process into the sample feeding module and the nucleic acid amplification reaction module when the detection result of the nucleic acid detecting apparatus is positive, dissolve or flush positive residues in the flow channel and the amplification reaction chamber, and then remove the positive residues through the high-temperature fluid cleaning module.

7. The nucleic acid detecting apparatus according to claim 2, wherein the sample sending module includes a plurality of containers for containing respective types of liquids required for the pretreatment, a plurality of mixing wells required for the premixing, hoses connecting the plurality of containers and the plurality of mixing wells, and a plurality of peristaltic pumps for driving the flow of the liquids in the hoses; the multiple containers comprise reaction liquid pools, positive liquid pools, negative liquid pools and extraction liquid pools, and the multiple mixing pools comprise nucleic acid extraction pools, positive control pools, negative control pools and sample pools to be detected;

the residue removing module is used for injecting fluid with the temperature of more than 200 ℃ into the pipeline to be cleaned when the detection result of the nucleic acid detection equipment is positive, and the pipeline to be cleaned at least comprises: the nucleic acid extraction pool, the sample pool to be detected and a hose communicated with the nucleic acid extraction pool and the sample pool are arranged on the sample conveying module, and the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber are arranged on the sample conveying module.

8. The nucleic acid detection apparatus according to claim 3, wherein the high-temperature fluid purging module is adapted to inject a fluid having a temperature of 200 ℃ or higher into the pipe to be purged in a high-pressure pulse manner;

the nucleic acid detection equipment also comprises a gas injection module, wherein the gas injection module is communicated with the sample delivery module and is used for injecting gas into a flow channel of the sample delivery module through an injection port, and each injection port is provided with a valve; the valve is suitable for closing the flow channel of the sample sending module and opening the air channel of the gas injection module so as to allow gas to be injected into the flow channel of the sample sending module; or opening the flow channel of the sample sending module and closing the air channel of the gas injection module to allow the liquid in the flow channel of the sample sending module to pass through;

the gas injection module and the high-temperature purging module share the same compressed gas cylinder, and the compressed gas cylinder is used for providing gas injected into the sample sending module.

9. The nucleic acid detecting apparatus according to claim 2, wherein the sample sending module and the nucleic acid amplification reaction module are integrated in a microfluidic chip.

10. The nucleic acid detection apparatus according to claim 2, wherein at least a part of the sections of the flow paths to be cleaned of the sample feeding module and the nucleic acid amplification reaction module are provided with heat insulating layers; the pipe to be cleaned at least comprises: the nucleic acid extraction pool of the sample sending module, the sample pool to be detected, a flow channel communicated with the sample pool, the amplification reaction chamber of the nucleic acid amplification reaction module and a flow channel communicated with the amplification reaction chamber.

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