A rapid multi-target nucleic acid quantitative analysis method and system, device, medium

Abstract

The application discloses a kind of fast multi-target nucleic acid quantitative analysis method and system, equipment, medium, belongs to molecular diagnosis and biological detection technical field.The method is injected into the fluid channel of reciprocating flow nucleic acid amplification system, and the reaction liquid containing nucleic acid sample, polymerase and fluorescent probe is completed by driving reaction liquid reciprocating flow in different temperature control interval amplification;After amplification ends, directly utilize temperature gradient region in system to collect the fluorescence signal of reaction liquid, and generate melting curve based on the corresponding relationship of fluorescence signal and temperature gradient, so as to seamlessly integrate amplification and melting analysis in the same system, realize the identification of multi-target nucleic acid.The application integrates melting curve analysis in reciprocating flow amplification process, does not increase additional temperature control step and sample transfer under the premise, significantly improves multi-target resolution capability, and has the advantages of rapid detection and high-resolution multi-target identification.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics and biological detection technology, and in particular to a rapid multi-target nucleic acid quantitative analysis method, system, equipment, and medium. Background Technology

[0002] Real-time quantitative PCR is an important technology in the field of molecular diagnostics. Rapid and multiplex target analysis is an important direction for its development. Conventional qPCR platforms achieve the temperature step changes required for nucleic acid amplification through the temperature-changing ability of Peltier, but the temperature-changing rate is relatively limited. It usually takes about 40 minutes to 1 hour to complete one nucleic acid amplification, which is difficult to meet the detection speed requirements of on-site nucleic acid testing.

[0003] To meet the demand for rapid on-site testing, isothermal amplification and CRISPER / Cas technologies have been gradually developed. Isothermal amplification technology offers fast detection speed and high sensitivity, but it has limitations in terms of multiplex detection capability and specificity. CRISPER / Cas technology has single-molecule detection capability, excellent detection specificity, and short detection time, but it has disadvantages in terms of the number of targets that can be detected simultaneously. PCR nucleic acid amplification technology remains the gold standard in the field of nucleic acid detection.

[0004] To address the slow amplification rate of PCR while retaining its advantages, existing technologies have proposed a rapid PCR nucleic acid amplification device based on reciprocating flow (see patent application CN202511720647.6). This device achieves rapid thermal cycling by driving the reaction solution to reciprocate between physically isolated high-temperature and low-temperature zones, effectively shortening the amplification time through a space-for-time tradeoff. However, existing reciprocating flow-based PCR methods still primarily rely on amplification curve analysis for multi-target detection. When the amplification efficiencies of different targets are similar or non-specific products are present, their multiple resolution capability is significantly limited, making it difficult to accurately distinguish multiple targets in the same reaction.

[0005] In summary, developing a nucleic acid detection method that combines high-speed amplification capability with high-resolution multi-target recognition capability based on a reciprocating flow rapid amplification platform has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] To achieve the above-mentioned objectives and other advantages of the present invention, a first objective of the present invention is to provide a rapid multi-target nucleic acid quantitative analysis method, comprising the following steps: A reaction solution containing a nucleic acid sample, polymerase, and fluorescent probe is injected into a fluid channel; wherein the fluid channel is located in a reciprocating flow cytometry nucleic acid amplification system and is used to connect at least two control zones with different temperatures in the system; The reaction solution is driven to flow back and forth between at least two control zones at different temperatures to perform nucleic acid amplification cycles. After amplification, the fluorescence signal of the reaction solution was collected as it flowed through a temperature gradient region. Based on the correspondence between the fluorescence signal and the temperature gradient, a melting curve is generated to identify multiple target nucleic acids in the sample.

[0007] Furthermore, the step of generating the melting curve includes: In the last amplification cycle, fluorescence images are acquired as the reaction solution enters the high-temperature control zone or moves from the low-temperature zone to the high-temperature zone, and the distribution of fluorescence intensity along the spatial position of the fluid channel is extracted. Based on the predetermined temperature-spatial location calibration curve, the fluorescence intensity-spatial distribution is converted into a fluorescence intensity-temperature curve, and then the melting peak is obtained through negative derivative analysis.

[0008] Furthermore, the temperature-spatial location calibration curve is obtained through the following steps: A temperature-sensitive fluorescent dye solution is injected into the fluid channel; The temperature control zone is set to gradually increase from low temperature to high temperature, and the fluorescence intensity of the fluorescent dye in the channel is recorded at different temperatures to establish a standard curve of temperature versus fluorescence intensity. Under the same optical conditions after the amplification of the reaction solution is completed, the fluorescence intensity at different positions in the channel is converted into the corresponding temperature value according to the standard curve, thus completing the temperature-spatial position calibration.

[0009] Furthermore, the inner diameter of the fluid channel is determined through simulation optimization, and the inner diameter ranges from 0.4 mm to 1.2 mm.

[0010] Furthermore, the at least two temperature control zones include a high-temperature zone and a low-temperature zone, and a gap is provided between the high-temperature zone and the low-temperature zone, the length of which ranges from 5mm to 30mm.

[0011] Furthermore, the duration of a single amplification cycle is 5 to 15 seconds.

[0012] Furthermore, the concentration of the polymerase ranges from 0.4 U / μL to 1.2 U / μL.

[0013] Furthermore, the fluorescent probe is a molecular beacon, and molecular beacons for different targets have different melting temperatures; by using the melting peaks at different characteristic temperatures in the melting curve, multiple target nucleic acids can be distinguished and quantified simultaneously.

[0014] A second objective of this invention is to provide a rapid multi-target nucleic acid quantification analysis system, comprising: The temperature control module includes at least two independently controlled temperature control zones; A fluid drive module is used to drive the reaction liquid to flow back and forth between the temperature control zones; The capillary tube serves as the fluid channel, used to contain the reaction liquid and facilitate heat exchange; A fluorescence detection module is used to collect the fluorescence signal of the reaction solution in the capillary tube during the amplification process; A data processing module, configured to perform the analysis method steps as described in any one of claims 1-8.

[0015] Furthermore, the data processing module includes: A temperature calibration unit is used to establish and store the correspondence between spatial position and temperature within the capillary tube. An image analysis unit is used to extract the fluorescence intensity of the reaction solution distributed along the pipeline space at a specific time. A curve generation unit is used to convert the fluorescence intensity distribution into a melting curve based on the spatial location-temperature correspondence. The identification unit is used to identify the type and / or content of the target in the sample based on the characteristic peaks in the melting curve.

[0016] Furthermore, the inner diameter of the capillary tube is 0.6 mm, the temperature control zone includes a high-temperature zone and a low-temperature zone, and the gap between the high-temperature zone and the low-temperature zone is 10 mm.

[0017] Furthermore, the fluorescence detection module includes a light source and a camera for capturing the spatial fluorescence distribution of the entire or part of the capillary tube in a single imaging process under a single temperature field.

[0018] A third objective of the present invention is to provide a computer device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps of the above-described method.

[0019] A fourth objective of the present invention is to provide a computer-readable storage medium having a computer program stored thereon, wherein the computer program, when executed by a processor, implements the steps of the above-described method.

[0020] Compared with the prior art, the beneficial effects of the embodiments of the present invention are: This invention employs a reciprocating flow cytometry nucleic acid amplification system, driving the reaction solution to flow back and forth between physically separated temperature control zones. By utilizing a spatial temperature field instead of the traditional time-temperature gradient, it significantly improves the thermal cycling rate of nucleic acid amplification, reducing the amplification time to the minute level. Furthermore, unlike traditional reciprocating flow techniques that rely solely on the amplification curve for quantification, this invention further collects fluorescence signals from the reaction solution in the temperature gradient region after amplification and generates a melting curve. This design allows the method to overcome the bottleneck of multi-target resolution while inheriting the rapid amplification advantages of reciprocating flow, achieving a synergistic effect of rapid and multi-target amplification.

[0021] This invention utilizes the spatial positional differences of the reaction solution as it flows through a temperature gradient region after amplification to reflect the melting temperature change. Compared with traditional melting curve analysis, this invention does not require an additional melting temperature variation step and directly completes high-resolution melting analysis on the amplification platform. The melting curve generated by the correspondence between fluorescence signal and temperature gradient can clearly distinguish different targets with similar melting temperatures (Tm values), effectively avoiding interference from non-specific amplification products or primer dimers, and significantly improving the signal-to-noise ratio and specificity of multiplex detection.

[0022] When collecting fluorescence signals, this invention can acquire spatial fluorescence distribution containing complete temperature information in a single imaging process at a single moment when all or part of the reaction solution enters the high-temperature zone (or temperature gradient zone). This method avoids the complex timing control required by traditional real-time quantitative PCR, which requires collecting light signals once per cycle, and also eliminates the need for a high-precision variable-temperature spectroscopy detection module. This simplifies the complexity of the optical and control systems and facilitates the miniaturization and cost reduction of the instrument.

[0023] This invention does not change the biochemical reaction nature of PCR amplification, and retains the advantages of PCR technology such as high sensitivity, wide dynamic range and mature reagent system. The endpoint analysis method based on melting curve does not rely on the calculation of the cycle threshold of Ct value, is less affected by the fluctuation of amplification efficiency, and the result interpretation is more intuitive and stable, making it more suitable for on-site rapid detection and primary healthcare applications.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 Flowchart of a rapid multi-target nucleic acid quantification method; Figure 2 Flowchart for oscillating flow-based multi-target nucleic acid detection; Figure 3 To generate a flowchart of the melting curve; Figure 4 This is a schematic diagram of a fluorescence temperature calibration device. Figure 5 This is a schematic diagram of temperature fluorescence calibration; Figure 6 This is a schematic diagram of a multiphysics simulation model; Figure 7 Flowchart for obtaining temperature spatial location calibration curves; Figure 8 Flowchart for determining the structural parameters of nucleic acid amplification pipeline; Figure 9 Flowchart for determining the high and low temperature gap; Figure 10 Flowchart of the method for optimizing the formulation conditions of nucleic acid amplification reagents; Figure 11 Flowchart for nucleic acid amplification performance verification; Figure 12 Flowchart for melting temperature space calibration; Figure 13 Flowchart of multiplex nucleic acid testing capabilities; Figure 14 Block diagram of a rapid multi-target nucleic acid quantification analysis system; Figure 15 A schematic diagram of computer equipment; Figure 16 This is a schematic diagram of a computer-readable storage medium. Detailed Implementation

[0026] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0027] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0028] The drawing numbers in this application are only used to distinguish the steps in the scheme and are not used to limit the execution order of the steps. The specific execution order is as described in the specification.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] This invention proposes a nucleic acid PCR amplification and multi-target analysis method based on the alternating flow of samples between different temperature zones. This method combines the advantages of rapid amplification and multi-target analysis, solving the problem of weak multi-target detection capability in traditional reciprocating flow PCR nucleic acid detection methods. The specific scheme is as follows: Example 1 A rapid multi-target nucleic acid quantification method, such as Figure 1 , Figure 2 As shown, it includes the following steps: S100. Inject the reaction solution containing nucleic acid sample, polymerase and fluorescent probe into the fluid channel; wherein the fluid channel is set in the reciprocating flow cytometry nucleic acid amplification system and is used to connect at least two control zones with different temperatures in the system; S200: Drive the reaction solution to flow back and forth between at least two control zones with different temperatures to perform nucleic acid amplification cycle; S300. After amplification, the fluorescence signal of the reaction solution is collected as it flows through the temperature gradient region. S400. Based on the correspondence between the fluorescence signal and the temperature gradient, a melting curve is generated to identify multiple target nucleic acids in the sample.

[0031] In some embodiments, such as Figure 3 As shown, the step of generating the melting curve includes: S410. In the last amplification cycle, acquire fluorescence images of the reaction solution as it enters the high-temperature control zone or moves from the low-temperature zone to the high-temperature zone, and extract the distribution of fluorescence intensity along the spatial position of the fluid channel. S420. Based on the predetermined temperature spatial location calibration curve, the fluorescence intensity spatial distribution is converted into a fluorescence intensity temperature curve, and then the melting peak is obtained through negative derivative analysis.

[0032] This embodiment establishes a precise correspondence between spatial location and temperature within the amplification pipeline, providing a temperature benchmark for subsequent melting curve analysis.

[0033] Specifically, this method establishes a pre-established correspondence between spatial location and temperature within the amplification tube before nucleic acid amplification, such as... Figure 7 As shown, the temperature spatial location calibration curve is obtained through the following steps: S421. Inject a temperature-sensitive fluorescent dye solution into the fluid channel; This embodiment uses a temperature-sensitive fluorescent dye for temperature calibration. Optional dyes include Tamra, FITC, Cy3, and Cy5, with a preferred concentration range of 1 nM to 500 nM. In this embodiment, Tamra fluorescent dye is selected at a concentration of 100 nM.

[0034] Inject 50 μL of Tamra fluorescent dye solution into the amplification capillary tubing, ensuring the tubing is filled with dye solution and free of air bubbles.

[0035] S422. Set the temperature control zone to gradually increase from low temperature to high temperature, record the fluorescence intensity of the fluorescent dye in the channel at different temperatures, and establish a standard curve of temperature versus fluorescence intensity. Specifically, a temperature control program for the heating plate is set, starting at 50°C and gradually increasing to 95°C in 1°C increments. Each temperature step is maintained for a sufficient time (30 seconds in this embodiment) to ensure the temperature of the dye solution inside the tube reaches equilibrium with the set temperature of the heating plate. During the stabilization period of each temperature step, a fluorescence imaging system is used to acquire fluorescence images of the dye solution inside the tube, and the average fluorescence intensity is calculated. The principle of the temperature calibration device is as follows: Figure 4 As shown.

[0036] The average fluorescence intensity corresponding to each collected temperature point is fitted to establish a standard conversion curve between temperature and fluorescence intensity. For example... Figure 5 As shown, the curve exhibits a typical negative correlation, meaning that the fluorescence intensity gradually decreases as the temperature increases. This curve will be used to subsequently invert the fluorescence signal into a temperature value.

[0037] S423. Under the same optical conditions after the amplification of the reaction solution is completed, the fluorescence intensity at different positions in the channel is converted into the corresponding temperature value according to the standard curve to complete the temperature spatial position calibration.

[0038] In other embodiments, different fluorescent dyes and concentration combinations were selected for comparison. The following five calibration solutions were prepared: (1) FITC, 50 nM; (2) FITC, 200 nM; (3) Cy3, 100 nM; (4) Cy5, 100 nM; (5) Tamra, 500 nM. Temperature fluorescence intensity conversion curves for each dye solution were established according to the calibration method described above.

[0039] All dyes exhibit good temperature sensitivity and can be calibrated at temperature. Tamra and FITC show a more linear response in the 50℃-95℃ range, making them suitable for applications requiring precise temperature measurement. Cy3 and Cy5 exhibit higher sensitivity in the high-temperature region (>80℃), making them suitable for high-temperature melting analysis. Those skilled in the art can select appropriate fluorescent dyes and concentrations based on specific detection needs.

[0040] In some embodiments, before amplification, the following preferred parameters are determined by simulation: the inner diameter of the fluid channel is determined by simulation optimization, and the inner diameter ranges from 0.4 mm to 1.2 mm; the at least two temperature control zones include a high temperature zone and a low temperature zone, and a gap is provided between the high temperature zone and the low temperature zone, and the length of the gap ranges from 5 mm to 30 mm.

[0041] To achieve optimal amplification efficiency and melting curve resolution, this embodiment optimizes key structural parameters of the capillary circuit through multiphysics simulation. Specifically, given the same sample volume (25 μL in this embodiment), a smaller inner diameter and a longer liquid column result in a larger surface area in contact with the temperature zones, which is beneficial for rapid heating and the formation of a longer temperature gradient in space, thereby improving the temperature resolution of the melting curve. However, an excessively small inner diameter leads to a significant increase in liquid driving resistance, which is detrimental to the rapid switching of the sample between different temperature zones.

[0042] To evaluate the optimal inner diameter of the pipeline, a multiphysics simulation model was established based on a dual-temperature zone structure, such as... Figure 6 As shown in the figure. In the simulation, the pipe inner diameter was set between 0.4 mm and 1.2 mm. The simulation results are as follows. Figure 8 As shown, Figure 8 (a) shows the length of the liquid column and the length of the temperature gradient formed for the same volume of liquid under different inner diameter conditions; the smaller the inner diameter, the longer the liquid column. Figure 8 (b) shows the difference in temperature distribution at the center of the liquid column under different inner diameter conditions; Figure 8 (c) shows the change in the heating and cooling rates of the sample under different inner diameter conditions; the smaller the inner diameter, the faster the temperature change rate. Figure 8 (d) in the figure shows the driving resistance of the liquid column under different inner diameter conditions. The smaller the inner diameter, the greater the driving pressure required.

[0043] Based on the simulation results above, and taking into account both rapid temperature changes and achievable driving resistance, the preferred pipe inner diameter in this embodiment is 0.6 mm.

[0044] The gap between the heating blocks in the high and low temperature zones must meet two conditions: (1) at any time, the temperature of the inner wall of the pipeline in this zone should not be lower than 60°C to ensure that the polymerase is not deactivated in the working state; (2) when the reaction sample flows through the gap, its temperature must always be maintained between 60°C and 72°C to provide the appropriate temperature required for the extension step.

[0045] In the simulation, the gap between the two temperature zones was set to 5mm to 30mm, and the simulation model was established as follows: Figure 9 As shown in (a) above. The simulation results are as follows. Figure 9 As shown, Figure 9 (b) shows the temperature distribution in the gap region under different gap sizes; Figure 9 (c) shows the temperature distribution cloud map of the entire pipeline under different gap widths; Figure 9 (d) in the figure shows the temperature distribution of the sample inside the tube at the gap position during the oscillation of the liquid sample in two temperature ranges.

[0046] Based on the simulation results above, the preferred gap width between the two temperature zones in this embodiment is 10 mm.

[0047] In some embodiments, the duration of a single amplification cycle is 5 to 15 seconds.

[0048] Furthermore, the concentration of the polymerase ranges from 0.4 U / μL to 1.2 U / μL.

[0049] To achieve the optimal balance between rapid amplification and amplification efficiency, this embodiment optimized the amplification cycle time and polymerase concentration. The relationship between different single-step amplification times (3 seconds, 5 seconds, 7 seconds, 9 seconds, and 12 seconds) and the fluorescence value at the amplification endpoint was tested, and the results are as follows: Figure 10 As shown in (a) above. Experimental results show that a significant response in the endpoint fluorescence begins when the single cycle time reaches 7 seconds. Considering both the overall amplification time (approximately 4-5 minutes for 40 cycles) and amplification efficiency, the amplification time for a single cycle was determined to be 7 seconds in this embodiment.

[0050] To accommodate rapid amplification reactions, nucleic acid amplification reactions were tested at enzyme concentrations of 0.4 U / μL, 0.6 U / μL, 0.8 U / μL, 1.0 U / μL, and 1.2 U / μL, with a fixed single-cycle time of 7 seconds. The optimal enzyme concentration was determined by evaluating the endpoint fluorescence intensity. The evaluation results are shown below. Figure 10 As shown in (b) of the figure. The experimental results show that the fluorescence intensity reaches a plateau at an enzyme concentration of 1.0 U / μL, and further increasing the enzyme concentration has limited effect on improving the amplification efficiency. Therefore, the optimal enzyme concentration selected in this example is 1.0 U / μL.

[0051] After the above optimization, amplification experiments were conducted at different template concentrations (10-fold serial dilutions) and with a negative control. The nucleic acid amplification results obtained are as follows: Figure 11 As shown in (a) above, the linear relationship between Ct value and template concentration is as follows: Figure 11 As shown in (b) of the figure, the results show a good linear relationship (R²>0.99) over a dynamic range of 6 orders of magnitude, indicating that the method has excellent quantitative detection capability.

[0052] In some embodiments, the fluorescent probe is a molecular beacon, and different molecular beacons for different targets have different melting temperatures (Tm); by using the melting peaks at different characteristic temperatures in the melting curve, multiple target nucleic acids can be distinguished and quantified simultaneously.

[0053] Specifically, the nucleic acid melting analysis is performed as the amplification system moves from the low-temperature region to the high-temperature region. When the amplification system moves from the low-temperature region to the high-temperature region, a temperature gradient is formed at the boundary. At this moment, fluorescence images of the high-temperature region are acquired, and the temperature changes of the amplification system are analyzed.

[0054] The specific steps for melting temperature spatial calibration include: shaking the fluorescent solution used for temperature calibration (same as in step one) in the tubing; and acquiring a fluorescence image when the fluorescent solution has completely entered the high-temperature zone. The fluorescence intensity is then analyzed and compared with... Figure 5 The fluorescence calibration curve is used to obtain the correspondence between position and temperature within the pipeline. The principle is as follows: Figure 12 As shown in (a) above, the repeated measurement results are as follows: Figure 12 As shown in (b) above, the temperature fitting curve is as follows: Figure 12 As shown in (c) above. This method can accurately map each pixel on the capillary to its corresponding temperature value, with a resolution of 0.1℃-0.3℃.

[0055] This embodiment uses the detection of multiple targets of bacterial infection as an example to verify the ability of this method to distinguish different targets. A multiplex PCR system targeting three bacteria (Klebsiella pneumoniae KP, Acinetobacter baumannii AB, and Pseudomonas aeruginosa PA) was designed.

[0056] Specific molecular beacon probes were designed for three types of bacteria, and the amplification effects of the three targets were as follows: Figure 13 As shown in (a) above, the designed melting temperatures (Tm) of the probes are: KP probe Tm≈79℃, PA probe Tm≈75℃, and AB probe Tm≈64℃.

[0057] Prepare separate mixtures containing three nucleic acid target templates, corresponding molecular beacon probes, and PCR reagents, and inject them into capillary tubes. Perform oscillating flow PCR amplification under the optimized conditions described above, with 45 cycles, a single cycle time of 7 seconds, and an enzyme concentration of 1.0 U / μL. After the final cycle is completed, acquire fluorescence images at the moment when all samples have entered the high-temperature zone during the last cycle.

[0058] Extracting the fluorescence image at the moment when all samples enter the high-temperature zone during the last cycle, the fluorescence intensity is distributed along the spatial location (i.e., temperature) of the capillary, resulting in a smooth fluorescence intensity-temperature curve.

[0059] Taking the negative derivative (-dF / dT) of this curve yields the melting peak curve. The results show three distinct independent peaks near 64℃, 75℃, and 79℃, corresponding to the specific amplification products of the AB, PA, and KP targets, respectively. Figure 13 As shown in (b) to (d) in the figure. The single-sample control experiment shows that each peak appears only when its corresponding target is present, proving that this melting analysis method can effectively distinguish multiplex PCR products.

[0060] The multiplex test results obtained by applying this method to two clinical samples are as follows: Figure 13 (e) and Figure 13 As shown in (f) in the diagram. The results show that... Figure 13 PA and KP infection were present in sample (e). Figure 13 AB infection was found in sample (f), consistent with the clinical diagnosis.

[0061] Example 2 A rapid multi-target nucleic acid quantification analysis system 500, such as Figure 14 As shown, it includes: The temperature control module 510 includes at least two independently controlled temperature control zones; in this embodiment, the temperature control module includes a high temperature zone (95°C) and a low temperature zone (60°C), as well as a heat insulation gap (10mm) between the two.

[0062] The fluid drive module 520 is used to drive the reaction liquid to flow back and forth between the temperature control zones; The capillary tube 530 serves as the fluid channel, used to contain the reaction liquid and achieve heat exchange; its inner diameter is 0.6 mm, and its total length is determined according to the sample volume, approximately 90 mm in this embodiment, corresponding to a 25 μL sample.

[0063] The fluorescence detection module 540 is used to collect the fluorescence signal of the reaction solution in the capillary tube during the amplification process; In some preferred embodiments, the fluorescence detection module includes a light source and a camera for capturing the spatial fluorescence distribution of the entire or part of the capillary tube in a single temperature field in a single imaging process.

[0064] The data processing module 550 is configured to execute the analysis method steps described above. For a detailed description of the method, please refer to the corresponding description in the above method embodiments, which will not be repeated here.

[0065] In some embodiments, the data processing module includes: The temperature calibration unit is used to establish and store the correspondence between the spatial position and temperature within the capillary tube, i.e., temperature calibration data based on fluorescent dye. The image analysis unit is used to extract the fluorescence intensity of the reaction solution distributed along the pipeline space at a specific moment; specifically, it is used to extract the fluorescence intensity of the reaction solution distributed along the pipeline space when it enters the high-temperature zone in the last cycle. A curve generation unit is used to convert the fluorescence intensity distribution into a melting curve based on the spatial location-temperature correspondence. The identification unit is used to identify the type and / or content of the target in the sample based on the characteristic peaks in the melting curve. Specifically, it is used to identify the type and / or content of the target in the sample based on the position (Tm value) and peak height / peak area of ​​the characteristic peaks in the melting curve.

[0066] In some preferred embodiments, the inner diameter of the capillary tube is 0.6 mm, the temperature control zone includes a high-temperature zone and a low-temperature zone, and the gap between the high-temperature zone and the low-temperature zone is 10 mm.

[0067] Example 3 A computer device 600, such as Figure 15 As shown, the system includes a memory 610, a processor 620, and a computer program 630 stored in the memory and executable on the processor. When the processor executes the computer program, it implements the steps of a rapid multi-target nucleic acid quantification method. For a detailed description of the method, please refer to the corresponding description in the above method embodiments; it will not be repeated here.

[0068] Example 4 A computer-readable storage medium, such as Figure 16 As shown, a computer program is stored thereon, which, when executed by a processor, implements the steps of a rapid multi-target nucleic acid quantitative analysis method. For a detailed description of the method, please refer to the corresponding description in the above method embodiments, and will not be repeated here.

[0069] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0070] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0071] The apparatus, computer device, and non-volatile computer storage medium and method provided in the embodiments of this specification are corresponding. Therefore, the apparatus, computer device, and non-volatile computer storage medium also have similar beneficial technical effects as the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding apparatus, computer device, and non-volatile computer storage medium will not be repeated here.

[0072] Those skilled in the art will also know that, besides implementing the controller in the form of purely computer-readable program code, the same functions can be achieved by logically programming the method steps, making the controller take the form of logic gates, switches, application-specific integrated circuits (ASICs), programmable logic controllers (PLCs), and embedded microcontrollers. Therefore, such a controller can be considered a hardware component, and the devices included within it for implementing various functions can also be considered structures within that hardware component. Alternatively, the devices for implementing various functions can be considered as both software units implementing the method and structures within a hardware component.

[0073] The systems, apparatuses, or units described in the above embodiments can be implemented by computer chips or physical entities, or by products with certain functions. For ease of description, the above apparatuses are described separately as various units based on their functions. Of course, when implementing one or more embodiments of this specification, the functions of each unit can be implemented in one or more software and / or hardware.

[0074] Those skilled in the art will understand that the embodiments of this specification can be provided as methods, systems, or computer program products. Therefore, the embodiments of this specification can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the embodiments of this specification can take the form of a computer program product implemented on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0075] This specification is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this specification. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart illustrations and / or block diagrams. Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0076] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0077] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0078] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0079] This specification may be described in the general context of computer-executable instructions, such as program units, that are executed by a computer. Generally, program units include routines, programs, objects, components, data structures, etc., that perform a specific task or implement a specific abstract data type. This specification may also be practiced in distributed computing environments, where tasks are performed by remote processing devices connected via a communication network. In distributed computing environments, program units may reside in local and remote computer storage media, including storage devices.

[0080] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0081] The above description is merely an embodiment of this specification and is not intended to limit the scope of one or more embodiments of this specification. Various modifications and variations can be made to one or more embodiments of this specification by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of one or more embodiments of this specification should be included within the scope of the claims of one or more embodiments of this specification.

Claims

1. A rapid multi-target nucleic acid quantitative analysis method, characterized in that, Includes the following steps: A reaction solution containing a nucleic acid sample, polymerase, and fluorescent probe is injected into a fluid channel; wherein the fluid channel is located in a reciprocating flow cytometry nucleic acid amplification system and is used to connect at least two control zones with different temperatures in the system; The reaction solution is driven to flow back and forth between at least two control zones at different temperatures to perform nucleic acid amplification cycles. After amplification, the fluorescence signal of the reaction solution was collected as it flowed through a temperature gradient region. Based on the correspondence between the fluorescence signal and the temperature gradient, a melting curve is generated to identify multiple target nucleic acids in the sample.

2. The rapid multi-target nucleic acid quantitative analysis method as described in claim 1, characterized in that, The step of generating the melting curve includes: In the last amplification cycle, fluorescence images are acquired as the reaction solution enters the high-temperature control zone or moves from the low-temperature zone to the high-temperature zone, and the distribution of fluorescence intensity along the spatial position of the fluid channel is extracted. Based on the predetermined temperature-spatial location calibration curve, the fluorescence intensity-spatial distribution is converted into a fluorescence intensity-temperature curve, and then the melting peak is obtained through negative derivative analysis.

3. The rapid multi-target nucleic acid quantitative analysis method as described in claim 2, characterized in that, The temperature-spatial location calibration curve is obtained through the following steps: A temperature-sensitive fluorescent dye solution is injected into the fluid channel; The temperature control zone is set to gradually increase from low temperature to high temperature, and the fluorescence intensity of the fluorescent dye in the channel is recorded at different temperatures to establish a standard curve of temperature versus fluorescence intensity. Under the same optical conditions after the amplification of the reaction solution is completed, the fluorescence intensity at different positions in the channel is converted into the corresponding temperature value according to the standard curve, thus completing the temperature-spatial position calibration.

4. The rapid multi-target nucleic acid quantitative analysis method as described in claim 1, characterized in that, The inner diameter of the fluid channel was determined through simulation optimization, and the inner diameter range is from 0.4 mm to 1.2 mm.

5. The rapid multi-target nucleic acid quantitative analysis method as described in claim 1, characterized in that, The at least two temperature control zones include a high-temperature zone and a low-temperature zone, and a gap is provided between the high-temperature zone and the low-temperature zone, the length of which ranges from 5 mm to 30 mm.

6. The rapid multi-target nucleic acid quantitative analysis method as described in claim 1, characterized in that, The duration of a single amplification cycle is 5 to 15 seconds.

7. The rapid multi-target nucleic acid quantitative analysis method as described in claim 1, characterized in that, The concentration range of the polymerase is 0.4 U / μL to 1.2 U / μL.

8. The rapid multi-target nucleic acid quantitative analysis method as described in claim 1, characterized in that, The fluorescent probe is a molecular beacon, and different targets have different melting temperatures. By using the melting peaks at different characteristic temperatures in the melting curve, multiple target nucleic acids can be distinguished and quantified simultaneously.

9. A rapid multi-target nucleic acid quantification analysis system, characterized in that, include: The temperature control module includes at least two independently controlled temperature control zones; A fluid drive module is used to drive the reaction liquid to flow back and forth between the temperature control zones; The capillary tube serves as the fluid channel, used to contain the reaction liquid and facilitate heat exchange; A fluorescence detection module is used to collect the fluorescence signal of the reaction solution in the capillary tube during the amplification process; A data processing module, configured to perform the analysis method steps as described in any one of claims 1-8.

10. The rapid multi-target nucleic acid quantitative analysis system as described in claim 9, characterized in that, The data processing module includes: A temperature calibration unit is used to establish and store the correspondence between spatial position and temperature within the capillary tube. An image analysis unit is used to extract the fluorescence intensity of the reaction solution distributed along the pipeline space at a specific time. A curve generation unit is used to convert the fluorescence intensity distribution into a melting curve based on the spatial location-temperature correspondence. The identification unit is used to identify the type and / or content of the target in the sample based on the characteristic peaks in the melting curve.

11. The rapid multi-target nucleic acid quantitative analysis system as described in claim 9, characterized in that, The inner diameter of the capillary tube is 0.6 mm, and the temperature control zone includes a high-temperature zone and a low-temperature zone, with a gap of 10 mm between the high-temperature zone and the low-temperature zone.

12. The rapid multi-target nucleic acid quantitative analysis system as described in claim 9, characterized in that, The fluorescence detection module includes a light source and a camera, used to capture the spatial fluorescence distribution of the entire or part of the capillary tube under a single temperature field in a single imaging process.

13. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that, When the processor executes the computer program, it implements the steps of the method as described in any one of claims 1 to 8.

14. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the steps of the method as described in any one of claims 1 to 8.

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