Hot air type qPCR instrument, multichannel detection optical system and detection method

Abstract

The invention discloses a hot air type qPCR instrument, a multi-channel detection optical system and a detection method, and relates to the field of molecular biological detection. The problems that an optical detection system adopted by an existing qPCR instrument mostly depends on a single-channel or double-channel design, sample hole sites need to be switched one by one and detected in a time-sharing mode in the detection process, namely, the efficiency is low in the face of a high-throughput requirement scene are solved, and the system combines multiple LED light sources with an optical filter to generate excitation wavelengths to form a light source module; after the multi-stage dichroscope is utilized to form a light combination module, the light combination module is transmitted to a reaction hole through one branch of the forked optical fiber to excite fluorescent substances, a fluorescent signal is collected through a common end of the forked optical fiber, the other branch of the forked optical fiber outputs a fluorescent signal, and the fluorescent signal is split by the reflection grating to form a light splitting module. And the array CMOS is combined with a spectrum unmixing algorithm to form a detection module for synchronous receiving, so that multi-channel simultaneous high-sensitivity detection is realized. The method is also suitable for the field of multi-pathogen detection and gene expression analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and in particular to a hot air qPCR instrument, a multi-channel detection optical system and a detection method. Background Art

[0002] Amid the booming development of modern biotechnology and precision medicine, real-time fluorescence quantitative PCR (qPCR) technology, which enables dynamic monitoring of DNA amplification through fluorescent probe labeling, is a core technology in precision medicine and life science research. It has become an indispensable key tool in many fields such as molecular biology research, clinical diagnosis, and epidemic prevention and control.

[0003] However, the optical detection systems used in current traditional qPCR instruments have gradually exposed a series of technical shortcomings that need to be addressed urgently.

[0004] From a throughput perspective, traditional architectures rely on single- or dual-channel designs, requiring sample well switching and time-sharing testing during the testing process. This makes them inadequate for high-throughput scenarios. For example, in large-scale infectious disease screening and initial sample screening for gene sequencing, testing a small number of samples is manageable, but testing hundreds or even thousands of samples is time-consuming and extremely inefficient, significantly hindering subsequent scientific research progress and clinical feedback.

[0005] Focusing on signal processing accuracy, existing technologies have obvious defects in the separation of excitation light and fluorescence signals. Ordinary filters are difficult to deeply block excitation light, and residual excitation light mixes into the fluorescence signal, significantly increasing background noise and greatly limiting detection sensitivity. For trace nucleic acid samples with low copy numbers, false negative results often occur. In addition, with the increase in the number of fluorescent markers, traditional spectral unmixing algorithms are complex and have poor accuracy. The signals of each fluorescence channel interfere with each other and are difficult to accurately separate and quantify, resulting in significant errors in gene quantification results, which cannot provide a reliable basis for scientific research and diagnosis.

[0006] Finally, the thermal cycling system and optical detection work poorly together. Traditional water bath or metal block heating temperature control methods suffer from slow ramp rates, poor temperature uniformity, and lengthy thermal cycling cycles, ultimately impacting the accuracy and timeliness of qPCR testing. Therefore, innovative solutions are urgently needed to overcome these bottlenecks. Summary of the Invention The present invention aims to address the problems of optical detection systems used in existing qPCR instruments, which mostly rely on single-channel or dual-channel designs. The detection process requires switching sample wells one by one and time-sharing detection, which leads to low efficiency in high-throughput scenarios, as well as poor detection accuracy and smoothness of the collaborative operation between the thermal cycler system and optical detection. To solve the above technical problems, the present invention is implemented through the following technical solutions: Solution 1. The present invention proposes a hot air qPCR instrument, which includes a bottom plate, an optical module, a bifurcated optical fiber, a microlens assembly housing, a microfluidic chip, a microfluidic chip pressure plate, a top cover, an elastic pressure cover, a top cover tray, a microfluidic chip carrier, a PCR instrument light-transmitting part, a middle tray, an instrument support rod, a shock-absorbing foot, a signal transmission interface, and a power interface; The optical module is placed on the bottom plate, and the end of the bifurcated optical fiber outside the optical module is connected to the microlens group housing to converge the excitation light onto the microfluidic chip, which is pressed by the microfluidic chip pressure plate. The top cover is connected to the elastic pressure cover through an elastic element, and the top cover is supported by the top cover tray. The microfluidic chip carrier is tightly attached to the light-transmitting parts of the PCR instrument, and the light-transmitting parts of the PCR instrument are supported by the middle tray and connected to the instrument support rod. The entire instrument is supported by shock-absorbing feet. The signal transmission interface transmits fluorescence signals to communicate with the computer, and the optical module is powered by the power interface.

[0007] Solution 2: A multi-channel detection optical system for a hot air qPCR instrument, the multi-channel detection optical system being implemented based on the hot air qPCR instrument described in Solution 1, comprising a first group of LED light sources, a second group of LED light sources, a third group of LED light sources, and a fourth group of LED light sources; a first group of converging lenses, a second group of converging lenses, a third group of converging lenses, and a fourth group of converging lenses; a first group of narrow-band filters, a second group of narrow-band filters, a third group of narrow-band filters, and a fourth group of narrow-band filters; a first dichroic mirror, a second dichroic mirror, and a third dichroic mirror; a first group of microlenses and a second group of microlenses; a plane reflector, a protective glass, a first focusing lens, and a second focusing lens; a notch filter, a reflection grating, and an array CMOS sensor; The light emitted by the first group of LED light sources, the second group of LED light sources, the third group of LED light sources, and the fourth group of LED light sources is converted into parallel light by the first group of converging lenses, the second group of converging lenses, the third group of converging lenses, and the fourth group of narrow-band filters, and is converged to the right end of the bifurcated optical fiber by the first dichroic mirror, the second dichroic mirror, the third dichroic mirror, the plane reflector, and the light-combining converging lens. The bifurcated optical fiber transmits excitation light and fluorescence, the notch filter filters out interference from the excitation light, the reflective grating separates multi-color fluorescence, and the array CMOS sensor receives all fluorescence signals simultaneously. The array CMOS sensor is combined with a spectral unmixing algorithm to form a detection module for synchronous reception, thereby realizing multi-channel simultaneous high-sensitivity detection.

[0008] Furthermore, a preferred embodiment is provided, wherein the system further comprises a PCB board, a converging lens sleeve, a converging lens spacer, a light source module housing, a light source module pressure ring, a light combining module housing, a light combining lens pressure ring, The PCB board is fixed inside the light source module housing, and the first group of LED light sources, the second group of LED light sources, the third group of LED light sources, and the fourth group of LED light sources are welded on the heat dissipation substrate of the PCB board; a converging lens sleeve and a converging lens spacer are sequentially arranged in front of each group of LED light sources for installing the converging lens and adjusting the distance between the converging lens and the LED light source; the first group of narrow-band filters, the second group of narrow-band filters, the third group of narrow-band filters, and the fourth group of narrow-band filters are fixed to the front end of the converging lens sleeve by UV glue; the light source module pressing ring locks the front end of the housing through a thread or a snap structure, pressing the converging lens sleeve and the filter; the light combining module housing is docked with the light source module housing, and the first dichroic mirror, the second dichroic mirror, the third dichroic mirror and the plane reflector are installed inside, and the converging lens is fixed at the end by the light combining lens pressing ring; the right end of the forked optical fiber is fixed to the light combining module housing through an optical fiber connecting flange, and a ceramic sleeve is embedded in the flange to ensure that the optical fiber end face is accurately aligned with the focus of the light combining lens.

[0009] Furthermore, a preferred embodiment is provided, wherein the first group of LED light sources, the second group of LED light sources, the third group of LED light sources, and the fourth group of LED light sources, each group is composed of 4 high-power LED light sources with different peak wavelengths connected in parallel, the optical power of a single LED is ≥, its driving current is 100mA, and the voltage is 3.2V.

[0010] Furthermore, a preferred embodiment is provided, wherein the first dichroic mirror, the second dichroic mirror, and the third dichroic mirror are arranged in cascade in ascending order of excitation wavelength from 530 nm to 550 nm to 640 nm, and the first dichroic mirror, the second dichroic mirror, the third dichroic mirror and the plane reflector are installed respectively in order from short to long wavelength.

[0011] Furthermore, a preferred embodiment is provided, wherein the first group of micro lenses and the second group of micro lenses are composed of two pieces of H-K9L material, focal length 5mm, numerical aperture NA=0.4 aspheric lens, through the thickness tolerance The microlens group spacers adjust the lens spacing.

[0012] Furthermore, a preferred embodiment is provided, wherein the notch filter is embedded in the precision slot of the inner wall of the spectrometer module sleeve by the spacers on both sides, and the filter substrate is BK7 glass, plated The dielectric film has a transmittance of ≤0.01% in the excitation wavelength band and an average transmittance of ≥90% in the fluorescence emission band; the notch filter is fixed with a UV-curing adhesive with a refractive index of 1.50, and the edge is buffered by an elastic silicone ring.

[0013] Furthermore, a preferred embodiment is provided, wherein the reflection grating is a holographic grating with adjustable blaze angle, the substrate is a zero-expansion glass of 100 mm×50 mm×6 mm, and the line density is 600-1200 lines / mm.

[0014] Furthermore, a preferred embodiment is provided, wherein the array CMOS sensor realizes real-time demixing of four-channel fluorescence signals through FPGA.

[0015] Solution 3: A multi-channel optical detection method for a hot air qPCR instrument, the multi-channel optical detection method being implemented based on the multi-channel optical detection system of the hot air qPCR instrument described in Solution 2, the method comprising the following steps: Step 1: Unlock the microfluidic chip carrier, insert the microfluidic chip pre-filled with qPCR reaction solution, and add the reagents to be tested to each reaction well in turn. Mineral oil; constant pressure is applied to seal the reaction hole through the microfluidic chip pressure plate to ensure that there is no bubble interference in the optical path; Step 2: Start the hot air qPCR instrument and perform the system initialization procedure, including optical system power self-test, hot air circulation module preheating, and array CMOS sensor cooling module startup; verify the optical module's optical path alignment using the built-in calibration light source, and use the FPGA controller to test the stability of each group of LED light source drive currents; set the thermal cycle parameters through the human-computer interface, including pre-denaturation temperature of 95°C, denaturation temperature of 95°C, annealing temperature of 55-65°C, extension temperature of 72°C, as well as the duration of each stage and the number of cycles of 40; simultaneously configure the fluorescence acquisition trigger time, specifying that optical detection should start at the end of the annealing stage; Step 3: The control circuit drives four sets of LED light sources to emit light synchronously in a pulse mode with a duty cycle of 50% and a frequency of 5kHz. The excitation light is combined by a converging lens, a narrow-band filter, and a dichroic mirror. The light is then transmitted from the branch end of the bifurcated optical fiber to the common end. The light is focused into a light spot by a microlens group. The four sets of LED light sources evenly illuminate the samples in the four reaction wells. The single excitation time is 20ms. The LED junction temperature is monitored in real time by a thermocouple, and the drive current is dynamically adjusted to maintain the light intensity. Step 4: The stimulated fluorescence emitted by the sample is focused by the microlens group into the common end of the bifurcated optical fiber. The common end of the bifurcated optical fiber is then transmitted back to the spectrometer module, where it is filtered out by the notch filter to remove the residual excitation light. The reflection grating then splits the light to form a spectrum containing various fluorescence wavelengths. The fluorescence of different wavelengths is focused onto a preset pixel area of the array CMOS sensor. The array CMOS sensor synchronously collects spectral data from four positions. Each pixel generates an intensity signal according to a preset wavelength-position mapping table. The sensor synchronously collects spectral data from four positions with an exposure time of 50 ms, and simultaneously records the ambient temperature and dark current background value. Step 5: The signal processing unit preprocesses the raw spectral data, deducts the sensor background mean under no-light conditions, calibrates the non-uniformity coefficient of each pixel response based on a uniform light source, and uses wavelet transform to filter out high-frequency noise; the preprocessed data is subjected to principal component analysis to extract the first four principal components, and a linear fit is performed using the non-negative least squares method with a standard spectral library of 1000 sets of spectral data containing four sets of LED light sources. The signal intensity of each fluorescence channel is separated, and the initial concentration of the DNA template and the amplification efficiency are calculated; Step 6: The hot air fluorescence quantitative PCR instrument's rotating mechanism drives the microfluidic chip to rotate in the reaction chamber, switching the detection targets of the four wells. When the stepper motor drives the microfluidic chip carrier to rotate, an S-shaped acceleration and deceleration curve is used to reduce the impact of mechanical vibration on the optical path. By monitoring the light intensity of the optical fiber end face, fine-tuning is performed after the rotation is in place to ensure that the deviation between the center of the light spot and the reaction well is ≤ If the detection signal of a certain well position exceeds the tolerance for three consecutive times, the system will automatically mark the well as invalid and reallocate the sample to a spare well position; the detection data will be backed up in real time to the dual redundant storage module; Step 7: The hot air circulation module quickly and evenly heats the reaction wells at a wind speed of 5 m / s during the denaturation stage, and reduces the wind speed to 2 m / s during the annealing stage to improve temperature stability. The FPGA timing controller accurately synchronizes the temperature gradient switching with the fluorescence acquisition timing to ensure accurate synchronization of signal acquisition. The total duration of a single thermal cycle is ≤ 45 s. The unmixed fluorescence intensity, Ct value, and amplification curve are stored in real time in the local database. The four-channel fluorescence intensity change curve, amplification kinetics diagram, and concentration quantification results are displayed in real time through the human-computer interaction interface. A PDF format test report is generated, including the sample ID, test time, target gene Ct value, and melting curve, to complete the test.

[0016] The present invention is beneficial in that: The hot air qPCR instrument and multi-channel detection optical system and detection method described in the present invention use four-well synchronous detection. A single run can process four fluorescent markers on four samples, increasing detection efficiency by four times compared to a single channel, making it suitable for multi-index combined detection. The use of bifurcated optical fibers instead of traditional confocal optical paths has the advantages of a compact optical path and low energy loss. Four sets of LED excitation optical paths are provided for the four wells, resulting in low crosstalk between channels. Multi-channel simultaneous detection is efficient and truly achieves simultaneous detection.

[0017] The hot-air qPCR instrument, multi-channel detection optical system, and detection method described in the present invention achieve synchronous excitation and fluorescence signal acquisition of multiple wells, significantly improving detection efficiency. At the same time, they effectively reduce interference between excitation light and fluorescence signals, enhancing detection sensitivity and accuracy. In addition, they simplify the spectral unmixing algorithm, shorten the unmixing time, and improve unmixing accuracy. Finally, they optimize the thermal cycle system to achieve rapid and precise temperature control, effectively cooperating with the optical detection system, thereby reducing system complexity and cost, and providing a more efficient and reliable solution for the field of molecular biology detection.

[0018] The present invention is also applicable to the fields of multiple pathogen detection and gene expression analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an overall cross-sectional view of a hot air qPCR multi-channel detection system according to the present invention.

[0020] Figure 2 This is an optical path diagram of a hot air qPCR multi-channel detection system described in the present invention.

[0021] Figure 3 This is a side view of the light-transmitting parts of a hot-air qPCR multi-channel detection system described in the present invention.

[0022] Figure 4 This is a side view of the optical module in the hot air qPCR multi-channel detection system of the present invention. Figure 5 This is a partial cross-sectional view of an optical module in a hot air qPCR multi-channel detection system according to the present invention.

[0023] Figure 6 This is a flow chart of a hot air qPCR multi-channel detection optical system detection method described in the present invention.

[0024] Among them, there are base plate 1, optical module 2, bifurcated optical fiber 3, microlens group housing 4, microfluidic chip 5, microfluidic chip pressure plate 6, top cover 7, elastic pressure cover 8, top cover tray 9, microfluidic chip carrier 10, PCR instrument light-transmitting parts 11, middle tray 12, instrument support rod 13, shock-absorbing feet 14, signal transmission interface 15, and power interface 16.

[0025] A first group of LED light sources 101, a second group of LED light sources 102, a third group of LED light sources 103, and a fourth group of LED light sources 104; a first group of converging lenses 105, a second group of converging lenses 106, a third group of converging lenses 107, a fourth group of converging lenses 108, a light-combining converging lens 205, a first group of narrow-band filters 109, a second group of narrow-band filters 110, a third group of narrow-band filters 111, and a fourth group of narrow-band filters 112, a first dichroic mirror 201, a second dichroic mirror 202, a third dichroic mirror 203, a first group of microlenses 401, a second group of microlenses 402, a plane reflector 204, a protective glass 403, a first focusing lens 601, a second focusing lens 603, a notch filter 602, a reflection grating 604, and an array CMOS sensor 701. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the implementation methods of this application clearer, the technical solutions in the implementation methods of this application will be clearly and completely described below in combination with the drawings in the implementation methods of this application. Obviously, the described implementation methods are only part of the implementation methods of this application, not all of the implementation methods.

[0027] Implementation method 1, see Figure 1 and Figure 6 To illustrate this embodiment, a hot air qPCR instrument includes a base plate 1, an optical module 2, a bifurcated optical fiber 3, a microlens assembly housing 4, a microfluidic chip 5, a microfluidic chip pressure plate 6, a top cover 7, an elastic pressure cover 8, a top cover tray 9, a microfluidic chip carrier 10, a PCR instrument light-transmitting part 11, a middle tray 12, an instrument support rod 13, a shock-absorbing foot 14, a signal transmission interface 15, and a power interface 16.

[0028] The optical module 2 is placed on the base plate 1. The end of the bifurcated optical fiber 3 outside the optical module 2 is connected to the microlens group housing 4 to converge the excitation light onto the microfluidic chip 5, which is pressed by the microfluidic chip pressing plate 6. The top cover 7 is connected to the elastic pressure cover 8 through an elastic element. The top cover 7 is supported by the top cover tray 9. The microfluidic chip carrier 10 is in close contact with the PCR instrument light-transmitting part 11. The PCR instrument light-transmitting part 11 is supported by the middle tray 12 and connected to the instrument support rod 13. The entire instrument is supported by the shock-absorbing foot 14. The signal transmission interface 15 transmits the fluorescence signal to communicate with the computer. The optical module 2 is powered by the power interface 16.

[0029] Implementation method 2: Figure 2As shown, a multi-channel detection optical system of a hot air qPCR instrument includes a first group of LED light sources 101, a second group of LED light sources 102, a third group of LED light sources 103, and a fourth group of LED light sources 104 of different wavelengths, a first group of converging lenses 105, a second group of converging lenses 106, a third group of converging lenses 107, and a fourth group of converging lenses 108, a first group of narrow-band filters 109, a second group of narrow-band filters 110, a third group of narrow-band filters 111, and a fourth group of narrow-band filters 112, a first dichroic mirror 201, a second dichroic mirror 202, and a third dichroic mirror 203, a plane mirror 204, a light-combining converging lens 205, a bifurcated optical fiber 3, a first group of microlenses 401 and a second group of microlenses 402; a protective glass 403, a microfluidic chip 5, a first focusing lens 601, a notch filter 602, a second focusing lens 603, a reflection grating 604, and an array CMOS sensor 701.

[0030] The light emitted by the first group of LED light sources 101, the second group of LED light sources 102, the third group of LED light sources 103, and the fourth group of LED light sources 104 of different wavelengths is converted into parallel light by the first group of converging lenses 105, the second group of converging lenses 106, the third group of converging lenses 107, and the fourth group of converging lenses 108, and is then converged to the right end of the bifurcated optical fiber 3 by the first dichroic mirror 201, the second dichroic mirror 202, the third dichroic mirror 203, the plane reflector 204, and the light-combining converging lens 205 through the first group of narrow-band filters 109, the second group of narrow-band filters 110, the third group of narrow-band filters 111, and the fourth group of narrow-band filters 112. The bifurcated optical fiber 3 transmits excitation light and fluorescence, the notch filter 602 filters out interference from the excitation light, the reflection grating 604 separates multi-color fluorescence, and the array CMOS sensor 701 receives all fluorescence signals simultaneously.

[0031] Because the system's four channels have four independent, identical light source systems, this embodiment uses a single optical system as an example. Four LED light sources, a first group 101, a second group 102, a third group 103, and a fourth group 104, are soldered to a PCB heat sink. A first group 105, a second group 106, a third group 107, and a fourth group 108 are positioned in front of the first, second, third, and fourth groups 101, 102, 103, and 104 to convert the light emitted by the LEDs into parallel light. UV adhesive is used to adhere a first group 109, a second group 110, a third group 111, and a fourth group 112 of narrowband filters. The parallel light passes through the first, second, third, and fourth groups 111, 112, and is converted into excitation light of the corresponding wavelength. The first dichroic mirror 201, the second dichroic mirror 202, the third dichroic mirror 203 and the plane mirror 204 are installed in order from short to long wavelengths. The 470nm light is reflected by the plane mirror 204 and transmitted through the third dichroic mirror 203. The 530nm light is reflected by the third dichroic mirror 203. The two wavelengths of light are combined and transmitted through the second dichroic mirror 202. The 550nm light is reflected by the second dichroic mirror 202. The three wavelengths of light are combined and transmitted through the first dichroic mirror 201. The 640nm light is reflected by the first dichroic mirror 201. The four wavelengths of excitation light are combined and focused through the light combining and converging lens 205 into the right end of the bifurcated optical fiber 3. like Figure 5As shown, the PCB board is fixed inside the light source module housing, and the four LED light sources, the first group LED light source 101, the second group LED light source 102, the third group LED light source 103, and the fourth group LED light source 104, are soldered on the heat dissipation substrate of the PCB board; the first group LED light source 101, the second group LED light source 102, the third group LED light source 103, and the fourth group LED light source 104 are sequentially provided with a converging lens sleeve and a converging lens spacer for installing the first group converging lens 105, the second group converging lens 106, the third group converging lens 107, and the fourth group converging lens 108 and adjusting the distance between them and the first group LED light source 101, the second group LED light source 102, the third group LED light source 103, and the fourth group LED light source 104 to ensure the collimation of the light path; the first group narrowband filter 109, the second group of narrow-band filters 110, the third group of narrow-band filters 111, and the fourth group of narrow-band filters 112 are fixed to the front end of the converging lens sleeve through UV glue; the light source module pressure ring locks the front end of the shell through a thread or a snap-on structure, pressing the converging lens sleeve and the filters; the light combining module shell is docked with the light source module shell, and the cascaded first dichroic mirror 201, the second dichroic mirror 202, the third dichroic mirror 203 and the plane reflector 204 are installed inside, and the end is fixed to the light combining converging lens 205 through the light combining lens pressure ring; the right end of the forked optical fiber 3 is fixed to the light combining module shell through the optical fiber connection flange, and the ceramic sleeve is embedded in the flange to ensure that the optical fiber end face is accurately aligned with the focus of the light combining lens; each component is mechanically aligned through positioning holes or guide columns, the overall structure is compact, the optical path is stable, and it meets the requirements of multi-channel synchronous detection.

[0032] In this embodiment, the common end of the bifurcated optical fiber 3 is fixed by a fiber common end connector and connected to the first group of microlenses 401 and the second group of microlenses 402; the microlens group housing 4 is connected to the fiber common end housing by a thread or a positioning pin, and a microlens group spacer and a microlens group pressure ring are sequentially arranged inside to adjust the spacing and optical axis alignment of the first group of microlenses 401 and the second group of microlenses 402 to ensure that the excitation spot diameter is ≤ ; The microlens group pressure ring locks the first group of microlenses 401 and the second group of microlenses 402 through an elastic pressure cover to prevent displacement caused by mechanical vibration; the light-transmitting parts include a protective glass 403 and a PCR instrument light-transmitting part 11, and the protective glass 403 is sealed to the front end of the microlens group housing 4 by UV glue to form an optical path channel between the microfluidic chip 5; the PCR instrument light-transmitting part 11 is connected to the top of the microlens group housing 4 by screws, and its light-transmitting hole position is strictly aligned with the optical axis of the first group of microlenses 401 and the second group of microlenses 402; the overall structure realizes efficient transmission of excitation light and low-loss collection of fluorescence signals through precise mechanical matching and sealing design, and sets four identical excitation light sources, bifurcated optical fibers 3, and microlens groups to meet the requirements of synchronous detection of multiple holes.

[0033] In this embodiment, the splitting end of the forked optical fiber 3 is fixed to the splitting module sleeve through the optical fiber splitting end connector, and the first focusing lens 601, the second focusing lens 603, the notch filter 602, and the reflection grating 604 are arranged in sequence inside the splitting module sleeve; wherein, the first focusing lens 601 adjusts the distance between itself and the end face of the forked optical fiber 3 through the splitting lens spacer to ensure that the fluorescence signal is collimated as parallel light; the notch filter 602 is fixed in the splitting module sleeve, and the excitation light transmittance is ≤0.01%, which effectively filters out residual excitation light interference; the reflection grating 604 is fixed to the inner surface of the splitting module sleeve at an inclined angle and is coated with a multi-layer dielectric film to improve the splitting efficiency; the end of the splitting module sleeve is connected to the array CMOS sensor 701 through a threaded connection; the splitting module is wrapped by an aluminum alloy shell as a whole and is connected to the instrument body through a positioning pin to ensure the stability of the optical path and the ability to resist electromagnetic interference, thereby realizing high-sensitivity synchronous detection of multi-color fluorescence signals.

[0034] In this embodiment, the excitation light emitted from the common end of the bifurcated optical fiber 3 is converged by the first group of microlenses 401 and the second group of microlenses 402, passes through the protective glass 403 and is focused onto the microfluidic chip 5. The reagents on the microfluidic chip 5 emit corresponding fluorescence. The multiple fluorescence can be regarded as a point light source. The emitted fluorescence passes through the protective glass 403 and is converged by the first group of microlenses 401 and the second group of microlenses 402, entering the common end of the bifurcated optical fiber 3, and emitting a fluorescence signal from the left end of the bifurcated optical fiber 3. After the fluorescence signal is emitted from the left end of the bifurcated optical fiber 3, it is focused into parallel light by the first focusing lens 601, filtered out by the notch filter 602 to remove interference from the excitation light, and then converged by the second focusing lens 603 so that the fluorescence signal becomes a tiny light spot when it reaches the array CMOS sensor 701. During the process of converging the fluorescence signal by the second focusing lens 603, the reflection grating 604 causes the fluorescence signals of different wavelengths to be focused on different pixel points of the array CMOS sensor 701, achieving the effect of fluorescence spectroscopic detection.

[0035] In this embodiment, the housing of the optical module 2 includes but is not limited to 6061-T6 aluminum alloy material, whose thermal conductivity is not less than 167W / (m·K), effectively increasing the heat dissipation surface area; the housing wall thickness is 2-3mm, and additional copper heat dissipation fins are added to key areas (such as the LED light source installation position), which are tightly attached to the PCB heat dissipation substrate through thermal conductive silicone grease to ensure that the LED operating temperature is stable at 25℃±2℃; the housing surface is sandblasted and pre-treated, and a hard anodizing process is used to form a thickness of ≥ The aluminum oxide layer has a microhardness of ≥400 HV and shows no signs of corrosion after a 240-hour salt spray test. The optical system housing is further designed for electromagnetic compatibility. The inner wall of the fully enclosed cavity is coated with conductive paint. Combined with the aluminum alloy substrate, it achieves an electromagnetic shielding effectiveness of ≥60dB. Metal spring finger pads provide electrical continuity with the instrument's main base. The housing seams are sealed with laser welding, achieving an IP54 protection rating. This effectively blocks external dust, moisture, and electromagnetic interference, ensuring the stability of high-sensitivity fluorescence signal acquisition.

[0036] The first LED light source group 101, the second LED light source group 102, the third LED light source group 103, and the fourth LED light source group 104 are composed of four high-power LEDs with different peak wavelengths connected in parallel. The optical power of a single LED is ≥ , its driving current is 100mA, voltage is 3.2V. After four pieces are connected in parallel, the total luminous flux is measured at a distance of 10mm from the light source. The light source drive circuit uses a constant current pulse mode with a 50% duty cycle and an adjustable frequency of 1-10kHz. An FPGA timing controller synchronizes LED lighting with the exposure cycle of the CMOS sensor. A thermocouple is used to monitor the LED junction temperature in real time, dynamically adjusting the drive current and reducing light intensity fluctuations. In the LED array, a narrowband filter with a bandwidth of 5-10nm and a half-width (FWHM) of ≤8nm is installed at the front end of each LED light source through a precision slot. The filter substrate is fused quartz, and the surface is coated with an ion beam sputtered multilayer dielectric film. The center wavelength strictly matches the excitation spectrum peak of the fluorescent dyes corresponding to 490nm±5nm for FAM, 530nm±5nm for HEX, 580nm±5nm for ROX, and 640nm±5nm for Cy5. The filter tilt angle is 3 degrees ±0.5 degrees, and the optical path is calibrated using a six-axis adjustment frame to ensure that the excitation light is perpendicular to the dichroic mirror assembly, with a wavelength matching error of ≤±3nm.

[0037] The first dichroic mirror 201, the second dichroic mirror 202, and the third dichroic mirror 203 are arranged in cascade in ascending order of excitation wavelengths from 530 nm to 550 nm to 640 nm. The first dichroic mirror 201, the second dichroic mirror 202, the third dichroic mirror 203 and the plane reflector 204 are installed in order from short to long wavelengths. The 470 nm light is reflected by the plane emitting mirror and transmitted through the bottom third dichroic mirror 203. The 530 nm light is reflected by the bottom third dichroic mirror 203. The two wavelengths of light are combined and transmitted through the middle second dichroic mirror 202. The 550 nm light is reflected by the middle second dichroic mirror 202. The three wavelengths of light are combined and transmitted through the top dichroic mirror. The 640 nm light is reflected by the top first dichroic mirror 201. The four wavelengths of excitation light are combined and focused by the light combining and converging lens 205 into the right end of the bifurcated optical fiber 3. Each dichroic mirror substrate is 0.5 mm thick UV-grade fused silica with a surface coating. The dielectric film, the incident angle is controlled at 45 degrees ± 0.1 degrees, the lens inclination is adjusted by a high-precision rotating table, and the four-wavelength optical axis deviation is calibrated by a laser interferometer after light combination ≤ 50 .

[0038] The first group of micro lenses 401 and the second group of micro lenses 402 are composed of two pieces of H-K9L material, focal length 5mm, numerical aperture NA = 0.4 aspheric lens, through the thickness tolerance The micro lens group spacer adjusts the lens spacing; the optical fiber combining end and optical fiber splitting end use FC / APC optical fiber connectors, with 8° bevel polishing on the end face, return loss ≤-60dB, and an outer diameter of 2.5mm and an inner diameter of 125 The ceramic ferrule is connected to the optical combining module and the optical splitting module, with an insertion loss of ≤0.3dB.

[0039] The notch filter 602 is embedded in the precision slot on the inner wall of the spectrometer module sleeve by the spacers on both sides. The filter base material is BK7 glass, plated The dielectric film has a transmittance of ≤0.01% in the excitation wavelength band and an average transmittance of ≥90% in the fluorescence emission band; the filter is fixed with a UV-curing adhesive with a refractive index of 1.50, and the edge is buffered with an elastic silicone ring to avoid stress cracking caused by temperature changes.

[0040] The spectroscopic module described in this embodiment has a reflective grating 604 that is a holographic grating with adjustable blaze angle. The substrate is a 100mm×50mm×6mm zero-expansion glass with a line density of 600-1200 lines / mm. The blaze angle is optimized for the four fluorescence wavelengths of FAM emission fluorescence of 520nm, HEX emission fluorescence of 560nm, ROX emission fluorescence of 610nm, and Cy5 emission fluorescence of 670nm. The grating surface is coated with a 150nm gold film and a trapezoidal groove structure is formed by ion beam etching to ensure that the first-order diffraction efficiency is ≥85%. The grating is mounted on Invar steel with a thermal expansion coefficient of 1. On the optical splitter module pillar, the tilt angle is dynamically adjusted by a piezoelectric ceramic actuator to compensate for the spectral shift caused by temperature drift.

[0041] The array CMOS sensor 701 has a photosensitive area surface coated with Broadband AR coating, pixel size , full well capacity The cooling module uses a two-stage semiconductor cooler TEC with a temperature difference of ΔT=60℃. The coolant temperature of the liquid cooling circulation system is 20℃±0.1℃, which stabilizes the sensor operating temperature at -10℃±0.5℃. The dark current The sensor output signal is converted by a 14-bit ADC, and the four-channel fluorescence signal is demixed in real time through FPGA.

[0042] In this embodiment, it also includes circuits and software parts such as an LED control module, a power supply control module, a signal processing module, and a subsequent spectrum unmixing algorithm.

[0043] Implementation method three: Figure 6 As shown, a multi-channel optical detection method of a hot air qPCR instrument includes the following steps: Step 1: Unlock the microfluidic chip carrier 10 and insert the microfluidic chip 5 pre-filled with qPCR reaction solution. Add 10 μL of mixed solution containing DNA template, fluorescent probe, Taq enzyme, dNTPs and buffer to each reaction well in turn, and cover with 2 μL of mineral oil to prevent evaporation; apply constant pressure through the microfluidic chip pressure plate 6 to seal the reaction well to ensure that there is no bubble interference in the optical path.

[0044] Step 2: Start the hot air qPCR instrument and perform the system initialization procedure, including a self-test of the optical system power supply, preheating the hot air circulation module, and starting the cooling module of the array CMOS sensor 701. The optical system's optical path alignment is verified using the built-in calibration light source, and the FPGA controller is used to test the driving current stability of the first, second, third, and fourth LED light sources. Thermal cycle parameters are set through the human-computer interface, including a pre-denaturation temperature of 95°C, a denaturation temperature of 95°C, an annealing temperature of 55-65°C, an extension temperature of 72°C, and the duration of each stage, with a total of 40 cycles. The fluorescence acquisition trigger time is also configured, specifying that optical detection will start at the end of the annealing stage.

[0045] Step 3: The control circuit drives the first group of LED light sources 101, the second group of LED light sources 102, the third group of LED light sources 103, and the fourth group of LED light sources (FAM-490nm, HEX-530nm, ROX-580nm, Cy5-640nm) to emit light synchronously in a pulse mode with a duty cycle of 50% and a frequency of 5kHz; the excitation light passes through the first group of converging lenses 105, the second group of converging lenses 106, the third group of converging lenses 107, the fourth group of converging lenses 108, and the first group of narrow band filters 10 9. A second set of narrow-band filters 110, a third set of narrow-band filters 111, and a fourth set of narrow-band filters 112; a first dichroic mirror 201, a second dichroic mirror 202, and a third dichroic mirror 203; light is then combined by a light-combining lens 205 and transmitted from one branch end of the bifurcated optical fiber 3 to the common end. The light is then focused into a light spot by microlens groups 401 and 402. The four light source systems evenly illuminate the samples in the four reaction wells, with a single excitation time of 20 ms. The LED junction temperature is monitored in real time by a thermocouple, and the drive current is dynamically adjusted to maintain light intensity.

[0046] Step 4: The first group of microlenses 401 and the second group of microlenses 402 focus the stimulated emission of the sample into the common end of the bifurcated optical fiber 3. The common end of the bifurcated optical fiber 3 transmits the emission back to the spectrometer module, where it is filtered out by the notch filter 602 to remove the residual excitation light. The reflection grating 604 then splits the light to form a spectrum containing various fluorescence wavelengths. The fluorescence of different wavelengths is focused onto the preset pixel area of the array CMOS sensor 701. The array CMOS sensor 701 synchronously collects spectral data of four positions. Each pixel generates an intensity signal according to the preset wavelength-position mapping table. The sensor synchronously collects spectral data of four positions with an exposure time of 50 ms and simultaneously records the ambient temperature and dark current background value.

[0047] Step 5: The signal processing unit preprocesses the raw spectral data, deducts the sensor background mean under no-light conditions, calibrates the non-uniformity coefficient of each pixel response based on a uniform light source, and uses wavelet transform to filter out high-frequency noise. After preprocessing, the data is extracted using principal component analysis (PCA) to extract the first four principal components. A linear fit is performed using the non-negative least squares method (NNLS) with a pre-stored standard spectral library of 1000 sets of spectral data containing FAM, HEX, ROX, and Cy5. The signal intensity of each fluorescence channel is separated, and the initial concentration of the DNA template and the amplification efficiency are calculated.

[0048] Step 6: The hot air fluorescence quantitative PCR instrument's rotating mechanism drives the microfluidic chip 5 to rotate within the reaction chamber, switching the detection targets of the four wells. The stepper motor drives the microfluidic chip 5's carrier stage to rotate using an S-shaped acceleration and deceleration curve to reduce the impact of mechanical vibration on the optical path. After the rotation is in place, fine-tuning is performed by monitoring the optical fiber end-face light intensity to ensure that the deviation between the center of the light spot and the reaction well is ≤±50μm. If the detection signal of a well exceeds the tolerance three times in a row, the system automatically marks the well as invalid and reallocates the sample to a spare well. Test data is backed up in real time to dual-redundant storage modules to prevent data loss due to sudden power outages.

[0049] Step 7: The hot air circulation module rapidly and evenly heats the reaction wells at 95°C at a wind speed of 5m / s during the denaturation phase. During the annealing phase, the wind speed is reduced from 60°C to 2m / s to improve temperature stability. The FPGA timing controller precisely synchronizes the temperature gradient switching with the fluorescence acquisition timing, ensuring precise synchronization of signal acquisition. The total duration of a single thermal cycle is ≤45s. The unmixed fluorescence intensity, Ct value, and amplification curve are stored in real time in the local database. The four-channel fluorescence intensity change curve, amplification kinetics diagram, and concentration quantification results are displayed in real time through the human-computer interface. A PDF test report is generated, including the sample ID, test time, target gene Ct value, melting curve analysis, and quality control labels. USB or wireless transmission to external devices is supported.

[0050] Step 8. After completing the nucleic acid amplification experiment, first turn off the instrument power and wait for the equipment to completely stop operating. Virus testing-related waste must be transferred to a professional agency for disposal. Routine laboratory waste must be sorted and stored in a sharps container or medical waste container and then disinfected with UV irradiation for unified disposal. During the instrument maintenance phase, the amplification module should be wiped with 75% ethanol, the carrier should be treated with chlorine-containing disinfectant, and the heated lid calibration and fluorescence signal correction should be performed regularly.

[0051] Those skilled in the art will understand that the above description is only a preferred embodiment of the present invention, and the features described in the various embodiments and / or technical solutions of the present disclosure can be combined or combined in various ways, even if such a combination or combination is not explicitly described in the present disclosure. It is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0052] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications as fall within the scope of the present invention and its equivalents.

Claims

1. A hot air qPCR instrument, characterized in that: The hot air qPCR instrument comprises a bottom plate (1), an optical module (2), a bifurcated optical fiber (3), a microlens assembly housing (4), a microfluidic chip (5), a microfluidic chip pressure plate (6), a top cover (7), an elastic pressure cover (8), a top cover tray (9), a microfluidic chip carrier (10), a PCR instrument light-transmitting part (11), a middle tray (12), an instrument support rod (13), a shock-absorbing foot (14), a signal transmission interface (15), and a power interface (16); An optical module (2) is placed on the bottom plate (1). The end of the bifurcated optical fiber (3) outside the optical module (2) is connected to the microlens group housing (4) to converge the excitation light to the microfluidic chip (5), which is pressed by the microfluidic chip pressing plate (6). The top cover (7) is connected to the elastic pressing cover (8) through an elastic element. The top cover (7) is supported by the top cover tray (9). The microfluidic chip carrier (10) is in close contact with the PCR instrument light-transmitting part (11). The PCR instrument light-transmitting part (11) is supported by the middle tray (12) and connected to the instrument support rod (13). The instrument as a whole is supported by a shock-absorbing foot (14). The signal transmission interface (15) transmits the fluorescence signal to communicate with the computer. The optical module (2) is powered by the power interface (16).

2. A multi-channel detection optical system for a hot air qPCR instrument, characterized in that: The multi-channel detection optical system is implemented based on the hot air qPCR instrument according to claim 1, and the system includes a first group of LED light sources (101), a second group of LED light sources (102), a third group of LED light sources (103), and a fourth group of LED light sources (104); a first group of converging lenses (105), a second group of converging lenses (106), a third group of converging lenses (107), a fourth group of converging lenses (108), and a light combining converging lens (205); a first group of narrow-band filters (109), A second group of narrowband filters (110), a third group of narrowband filters (111), and a fourth group of narrowband filters (112); a first dichroic mirror (201), a second dichroic mirror (202), and a third dichroic mirror (203); a first group of microlenses (401), and a second group of microlenses (402); a protective glass (403), a first focusing lens (601), and a second focusing lens (603); a notch filter (602), a reflection grating (604), and an array CMOS sensor (701); Light emitted by the first group of LED light sources (101), the second group of LED light sources (102), the third group of LED light sources (103), and the fourth group of LED light sources (104) is converted into parallel light by passing through the first group of converging lenses (105), the second group of converging lenses (106), the third group of converging lenses (107), and the fourth group of converging lenses (108), and is then passed through the first group of narrow-band filters (109), the second group of narrow-band filters (110), the third group of narrow-band filters (111), and the fourth group of narrow-band filters (112) and the first dichroic mirror (201). The second dichroic mirror (202), the third dichroic mirror (203), the plane reflector (204) and the light-combining converging lens (205) converge to the right end of the bifurcated optical fiber (3); the bifurcated optical fiber (3) transmits excitation light and fluorescence; the notch filter (602) filters out the interference of the excitation light; the reflective grating (604) separates the multi-color fluorescence; the array CMOS sensor (701) receives all the fluorescence signals simultaneously; the array CMOS sensor (701) is combined with a spectral unmixing algorithm to form a detection module for synchronous reception, thereby realizing multi-channel simultaneous high-sensitivity detection.

3. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The system also includes a PCB board, a converging lens sleeve, a converging lens spacer, a light source module housing, a light source module pressure ring, a light combining module housing, and a light combining lens pressure ring; The PCB board is fixed inside the light source module housing, and the first group of LED light sources (101), the second group of LED light sources (102), the third group of LED light sources (103), and the fourth group of LED light sources (104) are welded on the heat dissipation substrate of the PCB board; a converging lens sleeve and a converging lens spacer are sequentially arranged in front of each group of LED light sources for installing the converging lens and adjusting the distance between the converging lens and the LED light source; the first group of narrow-band filters (109), the second group of narrow-band filters (110), the third group of narrow-band filters (111), and the fourth group of narrow-band filters (112) are fixed to the front end of the converging lens sleeve by UV glue; The light source module pressing ring locks the front end of the housing through a thread or a snap-fit structure, pressing the converging lens sleeve and the filter; the light combining module housing is docked with the light source module housing, and a first dichroic mirror (201), a second dichroic mirror (202), a third dichroic mirror (203) and a plane reflector (204) are installed inside, and the end is fixed with the light combining converging lens (205) through the light combining lens pressing ring; the right end of the bifurcated optical fiber (3) is fixed to the light combining module housing through an optical fiber connecting flange, and a ceramic sleeve is embedded in the flange to ensure that the optical fiber end face is accurately aligned with the focus of the light combining lens.

4. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The first group of LED light sources (101), the second group of LED light sources (102), the third group of LED light sources (103), and the fourth group of LED light sources (104) are each composed of four high-power LED light sources with different peak wavelengths connected in parallel, and the optical power of a single LED is , its driving current is 100mA and voltage is 3.2V.

5. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The first dichroic mirror (201), the second dichroic mirror (202), and the third dichroic mirror (203) are arranged in cascade in ascending order of excitation wavelengths from 530 nm to 550 nm to 640 nm, and the first dichroic mirror (201), the second dichroic mirror (202), the third dichroic mirror (203), and the plane reflector (204) are installed in order from short to long wavelengths.

6. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The first group of micro lenses (401) and the second group of micro lenses (402) are composed of two pieces of H-K9L material, aspheric lenses with a focal length of 5 mm and a numerical aperture NA=0.4, and are The microlens group spacers adjust the lens spacing.

7. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The notch filter (602) is embedded in the precision slot on the inner wall of the spectrometer module sleeve by the spacers on both sides. The filter substrate is BK7 glass, plated The dielectric film has a transmittance of ≤0.01% in the excitation wavelength band and an average transmittance of ≥90% in the fluorescence emission band; the notch filter (602) is fixed by a UV-curing adhesive with a refractive index of 1.50, and an elastic silicone ring is used for buffering the edge.

8. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The reflection grating (604) is a holographic grating with adjustable blaze angle, a substrate of 100 mm×50 mm×6 mm zero expansion glass, and a line density of 600-1200 lines / mm.

9. The multi-channel detection optical system of the hot air qPCR instrument according to claim 2, characterized in that: The array CMOS sensor (701) realizes real-time demixing of four-channel fluorescence signals through FPGA.

10. A multi-channel optical detection method for a hot air qPCR instrument, characterized in that: The multi-channel detection optical method is implemented based on the multi-channel detection optical system of the hot air qPCR instrument according to claim 2, and the method comprises the following steps: Step 1: Unlock the microfluidic chip carrier (10), insert the microfluidic chip (5) pre-filled with qPCR reaction solution, add the reagents to be tested to each reaction well in turn, and cover Mineral oil; applying constant pressure to seal the reaction hole through the microfluidic chip pressure plate (6) to ensure that there is no bubble interference in the optical path; Step 2: Start the hot air qPCR instrument and execute the system initialization program, including self-test of the optical system power supply, preheating of the hot air circulation module, and starting of the array CMOS sensor (701) cooling module; verify the optical path alignment of the optical module (2) through the built-in calibration light source, and use the FPGA controller to detect the stability of the driving current of each group of LED light sources; set the thermal cycle parameters through the human-computer interaction interface, including the pre-denaturation temperature of 95°C, the denaturation temperature of 95°C, the annealing temperature of 55-65°C, the extension temperature of 72°C, and the duration of each stage, with a cycle number of 40 times; synchronously configure the fluorescence acquisition trigger time, and specify that the optical detection be started at the end of the annealing stage; Step 3: The control circuit drives four groups of LED light sources and emits light synchronously in a pulse mode with a duty cycle of 50% and a frequency of 5kHz; the excitation light is combined by a converging lens, a narrow-band filter and a dichroic mirror, and then transmitted from the branch end of the bifurcated optical fiber (3) to the common end, and is focused into a light spot by a microlens group. The four groups of LED light sources evenly illuminate the four reaction hole samples, and the single excitation time is 20ms; the LED junction temperature is monitored in real time by a thermocouple, and the driving current is dynamically adjusted to maintain the light intensity; Step 4: The fluorescence emitted by the sample under stimulation is focused by the microlens group and enters the common end of the bifurcated optical fiber (3). The common end of the bifurcated optical fiber (3) is transmitted back to the spectroscopic module, and the residual excitation light is filtered out by the notch filter (602) in turn. The reflection grating (604) splits the light to form a spectrum containing various fluorescence wavelengths. The fluorescence of different wavelengths is focused to the preset pixel area of the array CMOS sensor (701). The array CMOS sensor (701) synchronously collects the spectral data of the four holes. Each pixel generates an intensity signal according to the preset wavelength-position mapping table. The sensor synchronously collects the spectral data of the four holes with an exposure time of 50ms, and records the ambient temperature and dark current background value at the same time. Step 5: The signal processing unit preprocesses the raw spectral data, deducts the sensor background mean under no-light conditions, calibrates the non-uniformity coefficient of each pixel response based on a uniform light source, and uses wavelet transform to filter out high-frequency noise; the preprocessed data is subjected to principal component analysis to extract the first four principal components, and a linear fit is performed using the non-negative least squares method with a standard spectral library of 1000 sets of spectral data containing four sets of LED light sources. The signal intensity of each fluorescence channel is separated, and the initial concentration of the DNA template and the amplification efficiency are calculated; Step 6: The microfluidic chip (5) is driven to rotate in the reaction chamber by the rotating mechanism of the hot air fluorescence quantitative PCR instrument to switch the detection targets of the four wells; when the stepper motor drives the microfluidic chip carrier to rotate, an S-shaped acceleration and deceleration curve is used to reduce the influence of mechanical vibration on the light path; by monitoring the light intensity of the optical fiber end face, fine-tuning is performed after the rotation is in place to ensure that the deviation between the center of the light spot and the reaction well is correct. If the detection signal of a certain well position exceeds the tolerance for three consecutive times, the system will automatically mark the well as invalid and reallocate the sample to a spare well position; the detection data will be backed up in real time to the dual redundant storage module; Step 7: The hot air circulation module quickly and evenly heats the reaction wells at a wind speed of 5 m / s during the denaturation stage, and reduces the wind speed to 2 m / s during the annealing stage to improve temperature stability. The FPGA timing controller accurately synchronizes the temperature gradient switching with the fluorescence acquisition timing to ensure accurate synchronization of signal acquisition. The total duration of a single thermal cycle is ≤ 45 s. The unmixed fluorescence intensity, Ct value, and amplification curve are stored in real time in the local database. The four-channel fluorescence intensity change curve, amplification kinetics diagram, and concentration quantification results are displayed in real time through the human-computer interaction interface. A PDF format test report is generated, including the sample ID, test time, target gene Ct value, and melting curve, to complete the test.

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