WO2021243823A1 - 一种双通道实时荧光定量pcr仪光路系统及检测方法 - Google Patents

一种双通道实时荧光定量pcr仪光路系统及检测方法 Download PDF

Info

Publication number
WO2021243823A1
WO2021243823A1 PCT/CN2020/103159 CN2020103159W WO2021243823A1 WO 2021243823 A1 WO2021243823 A1 WO 2021243823A1 CN 2020103159 W CN2020103159 W CN 2020103159W WO 2021243823 A1 WO2021243823 A1 WO 2021243823A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorescence
dual
dichroic mirror
module
band
Prior art date
Application number
PCT/CN2020/103159
Other languages
English (en)
French (fr)
Inventor
娄凯
宋祺
高一博
唐昕
温维佳
Original Assignee
深圳市尚维高科有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市尚维高科有限公司 filed Critical 深圳市尚维高科有限公司
Publication of WO2021243823A1 publication Critical patent/WO2021243823A1/zh

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the invention relates to the technical field of biomedical detection, in particular to a dual-channel real-time fluorescent quantitative PCR instrument optical path system and a detection method.
  • PCR instrument is mainly based on polymerase chain reaction technology to achieve qualitative analysis of the results after a large number of specific DNA fragments in vitro are amplified in a short period of time.
  • Early PCR machines could only perform semi-quantitative and qualitative analysis using the "end-point method" for amplification first, which was not real-time, but also had disadvantages such as poor detection reproducibility and certain errors. Therefore, the real-time fluorescent quantitative PCR instrument came into being.
  • the present invention provides a dual-channel real-time fluorescent quantitative PCR instrument optical path system and detection method.
  • the optical path system combines the characteristics of miniaturization, integration, rapid response, and high sensitivity of microfluidic chip technology, which can further Broaden the field of application and better promote the development of multidisciplinary complementary advantages.
  • the optical path system of the dual-channel real-time fluorescent quantitative PCR instrument of the present invention has a compact structure and reduces light intensity loss at a transmission distance.
  • the detection method of the present invention realizes real-time synchronous imaging of fluorescence of different wavelengths, operates efficiently, and avoids interference between excitation light and fluorescence.
  • the first aspect of the present invention discloses a dual-channel real-time fluorescent quantitative PCR instrument optical path system, which includes a dual-color light excitation module 100, a microfluidic chip module 200, a dual-channel fluorescence detection module 300, and an image processing module 400;
  • the microfluidic chip module 200 is used to amplify the sample to be tested
  • the two-color light excitation module 100 is used to cause the sample to be tested to emit fluorescence, and to separate the excitation light from the excitation light source and the fluorescence from the sample to be tested;
  • the dual-channel fluorescence detection module 300 is used to realize the transmission of different wavelengths of fluorescence in different channels, adjust the light path of the emitted fluorescence of different wavelengths, so that the fluorescence of different wavelengths emitted by the sample to be tested is in the image capture device of the image processing module 400
  • the photosensitive surface of is on the same image surface and distributed in different spatial positions of the photosensitive surface;
  • the image processing module 400 is used for sensitizing the fluorescence and analyzing image data.
  • the two-color excitation module 100 includes two excitation light sources 111, 112, a short-band-pass dichroic mirror 120, a double-band-pass filter 130, and a double-band-pass dichroic mirror 140.
  • the two excitation light sources The excitation light wavelength is different;
  • the microfluidic chip module 200 includes a microfluidic chip 210 and a temperature rise and fall module 220, and the sample to be tested is clamped on the temperature rise and fall module 220;
  • the dual-channel fluorescence detection module 300 includes a first focusing lens 310, a long bandpass dichroic mirror 320, a single bandpass filter 331, 332, mirrors 341, 342, 343, and a second focusing lens 350;
  • the image processing module 400 includes an image capturing device 410;
  • a dual bandpass filter 130 and a dual bandpass dichroic mirror 140 are sequentially arranged between the short bandpass dichroic mirror 120 and the microfluidic chip 210;
  • a first focusing lens 310 is provided next to the dual band-pass dichroic mirror 140;
  • a long band-pass dichroic mirror (320) is provided between the focusing lens 310 and the first filter 331;
  • single-band-pass filters 331, 332 of corresponding wavelengths are respectively arranged;
  • a second focusing lens 350 is placed between the second mirror 342 and the image capturing device 410, and between the third mirror 343 and the image capturing device 410.
  • the excitation light emitted by each of the two excitation light sources 111, 112 is directly directed to a short-band-pass dichroic mirror 120, and the short-band-pass dichroic mirror 120 combines the two excitation lights, and the combined beam
  • the light passes through the dual band-pass filter 130 directly to the dual-band-pass dichroic mirror 140.
  • the light beam that directly hits the dual-band-pass dichroic mirror 140 is the incident light beam of the dual-band-pass dichroic mirror 140.
  • the double band-pass dichroic mirror 140 reflects the incident light beam and then vertically enters the sample to be tested, and then the fluorescence generated by the excitation of the sample to be tested passes through the double-band-pass dichroic mirror 140, the first focusing lens 310, After the long band pass dichroic mirror 320, the single band pass filters 331, 332, the mirrors 341, 342, 343, and the second focusing lens 350, they are converged on the photosensitive surface of the image capturing device 410.
  • the excitation light emitted by the first excitation light source 111 is transmitted through the short-bandpass dichroic mirror 120, and the excitation light emitted by the second excitation light source 112 is reflected by the short-bandpass dichroic mirror 120 , All are incident on the dual bandpass filter 130, after passing through the dual bandpass filter 130, are incident on the dual bandpass dichroic mirror 140.
  • the fluorescent light beam reflected by the second mirror 342 is incident on the lens 350;
  • the fluorescent light beam reflected by the first reflection mirror 341 is incident on the third reflection mirror 343, and the fluorescent light beam reflected by the third reflection mirror 343 is incident on the second focusing lens 350;
  • the light beam incident on the second focusing lens 350 is focused on the photosensitive surface of the image capturing device 410 through the second focusing lens 350.
  • the short-wavelength fluorescence passing through the first single-band pass filter 331, the first reflector 341, and the third reflector 343, and the long-wavelength fluorescence passing through the second single-band pass filter 332 and the second reflector 342 The fluorescence enters the lens 350 at a certain angle ⁇ .
  • the ⁇ >0 the value of ⁇ depends on the ratio of the focal lengths of the first lens 310 and the second lens 350.
  • the microfluidic chip 210 is on the front focal plane of the first focusing lens 310, the back focal plane of the first focusing lens 310 coincides with the front focal plane of the second focusing lens 350, and the image capturing device
  • the photosensitive surface 410 is on the back focal plane of the first focusing lens 310 to adjust the light path of the outgoing dual-wavelength fluorescent light so that the fluorescent light of different wavelengths is on the same image plane when it reaches the photosensitive surface 410 of the image capturing device.
  • the inclination angle of the reflecting mirror 342 and the third reflecting mirror 343 makes the fluorescence of different wavelengths be imaged on different positions of the photosensitive surface 410 of the image capturing device and there is no overlapping area.
  • the second aspect of the present invention discloses a method for real-time fluorescent quantitative PCR detection on a microfluidic chip, which adopts the dual-channel real-time fluorescent quantitative PCR instrument optical path system for detection, including the following steps:
  • step S100 a PCR reaction reagent is prepared, the sample to be tested is added to the PCR reaction reagent, and the PCR reaction reagent with the sample to be tested is added to the microfluidic chip.
  • step S200 the microfluidic chip is placed on the heating and cooling module, and the temperature program of the heating and cooling module is set to perform heating and cooling cycles, and fluorescence collection is performed.
  • step S300 the incident direction of the dual-color excitation light is directed toward the light-transmitting surface of the microfluidic chip, the fluorescent groups on the two fluorescent probes in the PCR reaction reagent emit fluorescence, and the outgoing fluorescence passes through the dual band-pass dichroic mirror , Through the lens convergence, and then through the long band-pass dichroic mirror to achieve different wavelength fluorescence sub-channel transmission, used in conjunction with the reflector, adjust the emitted dual-wavelength fluorescence light path, imaging on the photosensitive surface of the image capture device.
  • step S400 the fluorescent signal is collected, the fluorescent signal intensity curve is drawn, and the initial concentration of the target template is judged according to the curve to realize quantitative detection.
  • step of setting the temperature program of the heating and cooling module to perform heating and cooling cycles includes:
  • Heating cycle heating at the first temperature for the second time for denaturation, heating at the second temperature for the third time for annealing and extension, a total of multiple above-mentioned cycles are performed to achieve nucleic acid amplification;
  • Fluorescence collection is performed every time the second temperature heating is less than 2 seconds of the third time.
  • the optical path system includes a dual-color excitation module, a microfluidic chip module, a dual-channel fluorescence detection module and an image processing module; the dual-channel fluorescence detection The module is used to amplify the sample to be tested and collect fluorescence; the two-color excitation module is used to make the sample to be tested emit fluorescence, and separate the excitation light from the excitation light source and the fluorescence emitted from the sample to be tested; the microfluidic chip The module is used to realize the transmission of different wavelengths of fluorescence in different channels, and adjust the light path of the emitted fluorescence of different wavelengths so that the different wavelengths of fluorescence emitted by the sample to be tested are at different spatial positions of the photosensitive surface of the image capture device of the image processing module; The image processing module is used for sensitizing the fluorescence.
  • the invention uses dual channels to excite the fluorescence of the sample to be tested, and a single image capture device realizes real-time synchronous imaging of fluorescence of different wavelengths, which reduces the cost of the optical path system and the overall instrument; the optical path system is simple in design and compact in structure, and reduces the optical signal in the optical path system Transmission attenuation is also conducive to the miniaturization and modularization of the overall instrument; this optical path system also avoids the interference between excitation light and fluorescence, and improves the signal-to-noise ratio of the image capture device and the detection sensitivity of the instrument.
  • the present invention has the following beneficial technical effects:
  • the present invention uses dual channels to excite the fluorescence of the sample to be tested, and a single image capture device realizes real-time simultaneous imaging of fluorescence of different wavelengths, which reduces the cost of the optical path system and the overall instrument;
  • the optical path system of the dual-channel real-time fluorescent quantitative PCR instrument of the present invention has a compact structure and reduces the loss of light intensity in the transmission distance. This optical path system also avoids the interference between excitation light and fluorescence, and improves the signal-to-noise ratio of the image capture device. And the detection sensitivity of the instrument.
  • the detection method of the present invention realizes real-time synchronous imaging of fluorescence of different wavelengths, runs efficiently, and avoids interference between excitation light and fluorescence;
  • the optical path system of the present invention is simple in design and compact in structure, reduces the transmission attenuation of the optical signal in the optical path system, and is also conducive to the miniaturization and modularization of the overall instrument;
  • the optical path system of the present invention can further broaden the application field and better promote the development of multidisciplinary complementary advantages.
  • Figure 1 is a schematic diagram of the optical path system of the dual-channel real-time fluorescent quantitative PCR instrument of the present invention
  • Figure 2 is a structural diagram of the optical path system of the dual-channel real-time fluorescent quantitative PCR instrument of the present invention
  • Figure 3 is a graph of the amplification curve of the real-time fluorescent quantitative PCR detection of the microfluidic chip of the present invention
  • Fig. 4 is a flow chart of the real-time fluorescent quantitative PCR detection method of the microfluidic chip of the present invention.
  • the first aspect of the present invention provides a dual-channel real-time fluorescent quantitative PCR instrument optical path system, which can be applied to real-time fluorescent quantitative PCR detection on a microfluidic chip, and the optical path system can support at least two wavelengths of emission fluorescence detection.
  • the optical path system includes a dual-color excitation module 100, a microfluidic chip module 200, a dual-channel fluorescence detection module 300, and an image processing module 400.
  • the two-color excitation module 100 includes two excitation light sources 111 and 112, a short-bandpass dichroic mirror 120, a dual-bandpass filter 130, and a dual-bandpass dichroic mirror 140;
  • the microfluidic chip module 200 includes a microfluidic chip 210 and a temperature rise and fall module 220, and the sample to be tested is clamped on the temperature rise and fall module 220;
  • the dual-channel fluorescence detection module 300 includes a first focusing lens 310, a long band pass dichroic mirror 320, a first single band pass filter 331 and a second single band pass filter 332, a first reflector 341, The second reflecting mirror 342 and the third reflecting mirror 343, the second focusing lens 350;
  • the image processing module 400 includes an image capturing device 410, which may be a CMOS image capturing device.
  • a dual band pass filter 130 and a dual band pass dichroic mirror 140 are sequentially arranged between the short band pass dichroic mirror 120 and the microfluidic chip 210, and the excitation light emitted by the first excitation light source 111 passes through the The short-band-pass dichroic mirror 120 transmits and the excitation light emitted by the second excitation light source 112 is reflected by the short-band-pass dichroic mirror 120, and then is incident on the dual-band-pass filter 130, and passes through the dual-pass filter 130.
  • the bandpass filter 130 is incident on the dual bandpass dichroic mirror 140.
  • a first focusing lens 310 is provided next to the dual band pass dichroic mirror 140.
  • a long-band-pass dichroic mirror 320 is provided between the first focusing lens 310 and the first single-band-pass filter 331.
  • a long-band-pass dichroic mirror 320 is provided between the first focusing lens 310 and the second single-band-pass filter 332.
  • a first single-band-pass filter 331 of corresponding wavelength is arranged between the long-band-pass dichroic mirror 320 and the first mirror 341, and a first single-band-pass filter 331 of corresponding wavelength is arranged between the long-band-pass dichroic mirror 320 and the second mirror 342 There is a second single band pass filter 332 of the corresponding wavelength.
  • a lens 350 is placed between the second mirror 342 and the image capturing device 410, and between the third mirror 343 and the image capturing device 410.
  • the fluorescent light beam reflected by the second mirror 342 is incident on the lens 350.
  • the fluorescent light beam reflected by the first reflective mirror 341 is incident on the third reflective mirror 343, and the fluorescent light beam reflected by the third reflective mirror 343 is incident on the lens 350.
  • the light beam incident on the lens 350 is condensed on the photosensitive surface of the image capturing device 410 through the lens 350.
  • the first excitation light source 111 and the second excitation light source 112 are LED light sources of different wavelengths, which can be flexibly selected according to actual sample requirements.
  • the excitation light emitted by each of the two LED light sources is directly directed to a short-band-pass dichroic mirror 120.
  • the short-band-pass dichroic mirror 120 combines the two excitation lights, and the combined light is then filtered through a double-band-pass filter.
  • the sheet 130 directly hits the dual band-pass dichroic mirror 140.
  • the light beam directly hitting the dual-band-pass dichroic mirror 140 is the incident light beam of the dual-band-pass dichroic mirror 140.
  • the incident beam After the incident beam is turned, it is incident perpendicularly to the sample to be tested, and then the fluorescence generated from the sample to be tested passes through the double band pass dichroic mirror 140, the first focusing lens 310, the long band pass dichroic mirror 320, and the first single band in turn.
  • the pass filter 331 and the second single band pass filter 332, the first reflector 341, the second reflector 342 and the third reflector 343, and the lens 350 they are converged on the photosensitive surface of the image capturing device 410.
  • the center wavelengths of the two LED excitation light sources 111 and 112 in this embodiment are 470 nm and 625 nm, respectively, and the two emitted excitation lights directly hit the short-band pass dichroic mirror 120.
  • the short-bandpass dichroic mirror 120 with a cut-off wavelength of 500 nm combines the emitted light of the two LED excitation light sources to realize the real-time synchronous transmission of the dual-channel excitation light, and the real-time transmission of the beams of different excitation wavelengths through the shared channel.
  • the bandpass range of the dual bandpass filter 130 of this embodiment is 461-496nm and 621-653nm, and the dual bandpass filter 130 effectively filters out stray light around the center wavelength of the LED light sources 111 and 112.
  • the bandpass range of the dual bandpass dichroic mirror 140 of this embodiment is 500-540nm and 655-745nm.
  • the dual bandpass dichroic mirror 140 reflects the excitation light into the sample to be tested, and the fluorescence emitted by the sample It transmits through the dual band-pass dichroic mirror 140 to satisfy the separation of excitation light and fluorescence, and avoid transmission interference between the two.
  • the first focusing lens 310 condenses the emitted fluorescence transmitted through the dual band-pass dichroic mirror 140 to ensure that the width of the fluorescent beam does not exceed the long band-pass dichroic mirror 320, avoiding the loss of fluorescence signals and improving the collection efficiency.
  • the long-band-pass dichroic mirror 320 with a cut-off wavelength of 635nm realizes the channel transmission of fluorescence of different wavelengths. It is used in conjunction with the first mirror 341, the second mirror 342, and the third mirror 343 to adjust the light length of the emitted dual-wavelength fluorescence so that The fluorescence of different wavelengths is at the same Fourier back focal plane when it reaches the photosensitive surface of the image capturing device.
  • the dual-wavelength fluorescence emitted by the sample is on the same Fourier back focal plane when the photosensitive surface of the image capture device is exposed.
  • the center wavelength of the first single band pass filter 331 in this embodiment is 520 nm and the bandwidth is 40 nm; the center wavelength of the second single band pass filter 332 is 690 nm and the bandwidth is 50 nm. Both can effectively avoid stray light near the emitted fluorescence wavelength, and improve the signal-to-noise ratio and detection sensitivity.
  • the short-wavelength fluorescence passing through the first single-bandpass filter 331, the first mirror 341, and the third mirror 343, and the long-wavelength fluorescence passing through the second single-band-pass filter 332 and the second mirror 342 are clamped to a certain extent.
  • the angle ⁇ enters the lens 350, where ⁇ >0, and the value of ⁇ depends on the ratio of the focal lengths of the first lens 310 and the second lens 350.
  • the microfluidic chip 210 is on the front focal plane of the first focusing lens 310, and the rear focal plane of the first focusing lens 310 coincides with the front focal plane of the second focusing lens 350; the photosensitive surface 410 of the image capturing device is focused behind the first focusing lens 310 On the surface, adjust the light path of the outgoing dual-wavelength fluorescent light so that the fluorescent light of different wavelengths is on the same image plane when it reaches the photosensitive surface 410 of the image capture device. There are no overlapping areas on different positions of the photosensitive surface 410 of the device.
  • the second aspect of the present invention provides a method for real-time fluorescent quantitative PCR detection on a microfluidic chip, which adopts the aforementioned two-channel real-time fluorescent quantitative PCR instrument optical path system for detection, including the following steps, as shown in Fig. 4:
  • step S100 a PCR reaction reagent is prepared, the sample to be tested is added to the PCR reaction reagent, and the PCR reaction reagent with the sample to be tested is added to the microfluidic chip.
  • the sample to be tested can be a synthetic viral plasmid.
  • the synthetic viral plasmid is added to the PCR reaction reagent, and then the PCR reaction reagent with the viral nucleic acid plasmid is added To the microfluidic chip 210.
  • step S200 the microfluidic chip is placed on the heating and cooling module, and the temperature program of the heating and cooling module is set to perform heating and cooling cycles, and fluorescence collection is performed.
  • step S300 the incident direction of the dual-color excitation light is directed toward the light-transmitting surface of the microfluidic chip, and the fluorescent groups on the two fluorescent probes in the PCR reaction reagent emit outgoing fluorescence, and the outgoing fluorescence passes through the dual-band pass dichroic
  • the mirror converges through the lens, and then realizes the channel transmission of the fluorescence of different wavelengths through the long band-pass dichroic mirror. It is used in conjunction with the reflector to adjust the light length of the emitted dual-wavelength fluorescence, and the image is formed on the photosensitive surface of the image capture device.
  • step S400 the fluorescent signal is collected, the fluorescent signal intensity curve is drawn, and the initial concentration of the target template is judged according to the curve to realize quantitative detection.
  • the fluorescence signal of the collected graphics will gradually increase and remain unchanged after reaching a certain value.
  • the fluorescence intensity can be drawn to obtain a line of abscissa as time (Ct number, cycle number), and ordinate as fluorescence intensity According to this curve, the initial concentration of the target template can be judged, and the quantitative detection of the new coronavirus nucleic acid can be realized.
  • Fig. 1 it is shown that the above-mentioned light path system is used in the field of microfluidic chip technology with the temperature raising and lowering heating module to realize dual-channel real-time fluorescent quantitative PCR detection.
  • This embodiment can achieve specific detection of nucleic acids of various pathogenic microorganisms.
  • a novel coronavirus nucleic acid plasmid is specifically selected as the target template for detection, and a commercial detection kit is used for detection.
  • a PCR reaction reagent is prepared, and the synthesized novel coronavirus plasmid is added to the PCR reaction reagent according to the requirements of the commercial kit. Add the PCR reaction reagent with the new coronavirus nucleic acid plasmid to the microfluidic chip.
  • the light-transmitting surface of the microfluidic chip faces the incident direction of the two-color LED excitation light.
  • the fluorescent groups on the two fluorescent probes in the reagent emit fluorescence.
  • the band-pass dichroic mirror realizes the channel transmission of the fluorescence of different wavelengths, and is used in conjunction with the reflector to adjust the light length of the emitted dual-wavelength fluorescence, and the image is formed on the photosensitive surface of the image capture device.
  • the fluorescence signal of the collected graphics will gradually increase and remain unchanged after reaching a certain value.
  • the fluorescence intensity can be drawn to obtain a line of abscissa as time (Ct number, cycle number), and ordinate as fluorescence intensity
  • the initial concentration of the target template can be judged based on this curve, and the quantitative detection of nucleic acid of the novel coronavirus can be realized.
  • the present invention provides a dual-channel real-time fluorescence quantitative PCR instrument optical path system, including a dual-color excitation module, a microfluidic chip module, a dual-channel fluorescence detection module and an image processing module;
  • the dual-channel fluorescence detection module is used for Amplify the sample to be tested and collect fluorescence;
  • the dual-color excitation module is used to make the sample to be tested emit fluorescence, and to separate the excitation light emitted by the excitation light source from the fluorescence emitted by the sample to be tested;
  • the microfluidic chip module is used In order to realize the transmission of different wavelengths of fluorescence in different channels, adjust the light path of the emitted fluorescence of different wavelengths so that the different wavelengths of fluorescence emitted by the sample to be tested are on the same Fourier back focal plane on the photosensitive surface of the image capture device of the image processing module ;
  • the image processing module is used to sensitize the fluorescence.
  • the invention uses dual channels to excite the fluorescence of the sample to be tested, and a single image capture device realizes real-time synchronous imaging of fluorescence of different wavelengths, which reduces the cost of the optical path system and the overall instrument; the optical path system is simple in design and compact in structure, and reduces the optical signal in the optical path system Transmission attenuation is also conducive to the miniaturization and modularization of the overall instrument; this optical path system also avoids the interference between excitation light and fluorescence, and improves the signal-to-noise ratio of the image capture device and the detection sensitivity of the instrument.

Abstract

本发明提供一种双通道实时荧光定量PCR仪光路系统及检测方法，该光路系统包括双色光激发模块、微流控芯片模块、双通道荧光探测模块和图像处理模块；微流控芯片模块用于对待测样品扩增并采集荧光；双色光激发模块用于使待测样品出射荧光，并将激发光源出射的激发光与待测样品出射的荧光分离；双通道荧光探测模块用于实现不同波长荧光分通道传输,调整出射的不同波长荧光的光程。本发明采用双通道激发待测样品荧光，单台图像捕获装置实现不同波长荧光实时同步成像，降低光路系统及整体仪器成本；光路系统设计简单、结构紧凑，减少光路系统中的光信号传输衰减，有利于整体仪器小型化、模块化；避免激发光和荧光之间的干扰，提高图像捕获装置接收信噪比和仪器检测灵敏度。

Description

一种双通道实时荧光定量PCR仪光路系统及检测方法 技术领域
本发明涉及生物医学检测技术领域，具体涉及一种双通道实时荧光定量PCR仪光路系统及检测方法。
背景技术
生命科学仪器的不断发展为生物医学提供有力保障和有效研究工具。PCR仪作为一种广泛应用于生化分析、临床诊断、疾控筛查等多个领域的生命科学仪器，主要依据聚合酶链反应技术实现短时间内体外大量扩增特定DNA片段后结果定性分析。早期的PCR仪只能先扩增再采用“终点法”进行半定量、定性分析，这样既不具有实时性也存在检测重现性差有一定误差等弊端。因此，实时荧光定量PCR仪应运而生。其是指在PCR体系中结合荧光基团，利用荧光信号积累实时监测整个PCR进程，通过标准曲线进行定量分析。该技术不仅实现PCR从定性到定量的跨越，也具有特异性强、高效自动化等优势。随着基因科学和分子生物学等领域的发展，人们对实时荧光定量PCR仪提出更多的要求，大多趋向多色荧光通道发展，这样可以同时进行多重PCR及SNP等分析。但主要以多个独立检测光路通道并行，共用一套检测器的方式满足多通道实时荧光定量PCR仪的需求。这种方式需要对通道进行选择并逐一检测，这样一来将面临整体检测所需时间较长，探测器的口径大，光学系统结构所需空间较大，材料成本高等问题。
发明内容
为了解决上述问题，本发明提供了一种双通道实时荧光定量PCR仪光路系统及检测方法，该光路系统结合微流控芯片技术的微型化、集成化、 反应迅速、灵敏度高等特性，能够进一步地拓宽应用领域，更好地推动多学科优势互补型发展。本发明的双通道实时荧光定量PCR仪光路系统结构紧凑，减少传输距离光强损耗，本发明的检测方法实现了不同波长荧光实时同步成像，运转高效，避免激发光和荧光之间的干扰。
本发明采用如下的技术方案实现：
本发明的第一方面公开了一种双通道实时荧光定量PCR仪光路系统，包括双色光激发模块100、微流控芯片模块200、双通道荧光探测模块300和图像处理模块400；
所述微流控芯片模块200用于对待测样品扩增；
所述双色光激发模块100用于使待测样品出射荧光，并将激发光源出射的激发光与待测样品出射的荧光分离；
所述双通道荧光探测模块300用于实现不同波长荧光分通道传输,调整出射的不同波长荧光的光程，使得所述待测样品出射的不同波长荧光在所述图像处理模块400的图像捕获装置的感光面处于同一像面上并分布在感光面不同空间位置上；
所述图像处理模块400用于感光所述荧光，并对图像数据进行分析。
进一步的，所述双色激发模块100包括两个激发光源111，112、短带通二向色镜120、双带通滤光片130、双带通二向色镜140，所述两个激发光源的激发光波长不同；
所述微流控芯片模块200包括微流控芯片210和升降温模块220，待测样品夹持在所述升降温模块220上；
所述双通道荧光探测模块300包括第一聚焦透镜310，长带通二向色镜320，单带通滤光片331,332，反射镜341,342,343和第二聚焦透镜350；
所述图像处理模块400包括图像捕获装置410；
所述短带通二向色镜120和微流控芯片210之间依次设有双带通滤波片130和双带通二向色镜140；
紧邻所述双带通二向色镜140设有第一聚焦透镜310；
所述聚焦透镜310和第一滤波片331之间设有长带通二向色镜(320)；
在长带通二向色镜320与反射镜341，342之间分别设置有相应波长的单带通滤光片331，332；
在第二反射镜342与图像捕获装置410之间、第三反射镜343与图像捕获装置410之间放置第二聚焦透镜350。
进一步的，两个所述激发光源111，112各自出射的激发光分别直射到短带通二向色镜120，所述短带通二向色镜120将两束激发光合束，合束后的光再经双带通滤光片130直射到双带通二向色镜140，所述直射到双带通二向色镜140的光束是双带通二向色镜140的入射光束，所述双带通二向色镜140将所述入射光束反射后垂直入射到待测样品，之后从待测样品激发产生的荧光依次经所述双带通二向色镜140、第一聚焦透镜310、长带通二向色镜320、单带通滤光片331，332、反射镜341、342、343、第二聚焦透镜350后，汇聚于图像捕获装置410的感光面。
进一步的，第一激发光源111发出的激发光经所述短带通二向色镜120透射、以及所述第二激发光源112发出的激发光经所述短带通二向色镜120反射后，均入射到所述双带通滤光片130，经过所述双带通滤光片130后入射到所述双带通二向色镜140。
进一步的，经第二反射镜342反射的荧光光束入射到透镜350；
经第一反射镜341反射的荧光光束入射到第三反射镜343、再经第三反射镜343反射后的荧光光束入射到第二聚焦透镜350；
入射到第二聚焦透镜350的光束，经第二聚焦透镜350汇聚于图像捕获装置410的感光面。
进一步的，通过第一单带通滤光片331、第一反射镜341与第三反射镜343的短波长荧光，以及通过第二单带通滤光片332、第二反射镜342的长波长荧光以一定夹角α进入透镜350。
进一步的，所述α>0,所述α的值取决于第一透镜310与第二透镜350的焦距的比值。
进一步的，所述微流控芯片210在第一聚焦透镜310的前焦面上，所述第一聚焦透镜310的后焦面与第二聚焦透镜350的前焦面重合，所述图像捕获装置感光面410在所述第一聚焦透镜310的后焦面上，调整出射双波长荧光光程，使得不同波长荧光到达所述图像捕获装置感光面410时处于同一像面上，调节所述第二反射镜342和所述第三反射镜343的倾角使不同波长荧光成像在所述图像捕获装置感光面410的不同位置上并且没有重合区域。
本发明的第二方面公开了一种微流控芯片实时荧光定量PCR检测的方法，采用所述的双通道实时荧光定量PCR仪光路系统进行检测，包括如下步骤：
步骤S100，配制PCR反应试剂，将待测样本加入到所述PCR反应试剂中，并将所述带有待测样本的PCR反应试剂添加到微流控芯片中。
步骤S200,将所述微流控芯片置于升降温模块上，设置所述升降温模块的温度程序进行升降温循环，并进行荧光采集。
步骤S300,使双色激发光的入射方向朝向微流控芯片的透光面，PCR反应试剂中的两种荧光探针上的荧光基团发射荧光，所述出射荧光通过双带通二向色镜，经由透镜汇聚，再通过长带通二向色镜实现不同波长荧光分通道传输,与反射镜配合使用，调整出射双波长荧光光程，成像在图像捕获装置感光面。
步骤S400,采集荧光信号，绘制荧光信号强度曲线，根据所述曲线判断目标模板的初始浓度，实现定量检测。
进一步的，所述设置所述升降温模块的温度程序进行升降温循环的步骤包括：
第一温度加热第一时间，进行预变性；
升降温循环：第一温度加热第二时间进行变性，第二温度加热第三时间进行退火延伸，共进行多个上述循环，实现核酸扩增；
在每一次第二温度加热少于第三时间2秒的时候进行荧光采集。
综上所述，一种双通道实时荧光定量PCR仪光路系统和检测方法，该光路系统包括双色激发模块、微流控芯片模块、双通道荧光探测模块和图像处理模块；所述双通道荧光探测模块用于对待测样品扩增，并采集荧光；所述双色激发模块用于使待测样品出射荧光，并将激发光源出射的激发光与待测样品出射的荧光分离；所述微流控芯片模块用于实现不同波长荧光分通道传输,调整出射的不同波长荧光的光程使得所述待测样品出射的不同波长荧光在所述图像处理模块的图像捕获装置的感光面的不同空间位置处；所述图像处理模块用于感光所述荧光。
本发明采用双通道激发待测样品荧光，单台图像捕获装置实现不同波长荧光实时同步成像，降低了光路系统及整体仪器的成本；光路系统设计简单、结构紧凑，减少了光路系统中的光信号传输衰减，也有利于整体仪器小型化、模块化；此光路系统也避免了激发光和荧光之间的干扰，提高了图像捕获装置接收信噪比和仪器检测灵敏度。
与现有技术相比，本发明有如下有益的技术效果：
(1)、本发明采用双通道激发待测样品荧光，单台图像捕获装置实现不同波长荧光实时同步成像，降低了光路系统及整体仪器的成本；
(2)、本发明的双通道实时荧光定量PCR仪光路系统结构紧凑，减少传输距离光强损耗，此光路系统也避免了激发光和荧光之间的干扰，提高了图像捕获装置接收信噪比和仪器检测灵敏度。
(3)、本发明的检测方法实现了不同波长荧光实时同步成像，运转高效，避免激发光和荧光之间的干扰；
(4)、本发明的光路系统设计简单、结构紧凑，减少了光路系统中的光信号传输衰减，也有利于整体仪器小型化、模块化；
(5)、本发明光路系统结合微流控芯片技术的微型化、集成化、反应迅速、灵敏度高等特性，能够进一步地拓宽应用领域，更好地推动多学科优势互补型发展。
附图说明
图1是本发明的双通道实时荧光定量PCR仪光路系统示意图；
图2是本发明的双通道实时荧光定量PCR仪光路系统结构图；
图3是本发明的微流控芯片实时荧光定量PCR检测的扩增曲线图；
图4是本发明的微流控芯片实时荧光定量PCR检测的方法流程图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式并参照附图，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
本发明的第一方面提供了一种双通道实时荧光定量PCR仪光路系统，该光路系统可应用于微流控芯片实时荧光定量PCR检测，该光路系统至少可以支持两种波长出射荧光的检测。如图1所示，该光路系统包括双色激发模块100、微流控芯片模块200、双通道荧光探测模块300和图像处理模块400。具体的，所述双色激发模块100包括两个激发光源111和112、短带通二向色镜120、双带通滤光片130、双带通二向色镜140；
所述微流控芯片模块200包括微流控芯片210和升降温模块220，待测样品夹持在所述升降温模块220上；
所述双通道荧光探测模块300包括第一聚焦透镜310，长带通二向色镜320，第一单带通滤光片331和第二单带通滤光片332，第一反射镜341、第二反射镜342和第三反射镜343，第二聚焦透镜350；
所述图像处理模块400包括图像捕获装置410，该图像捕获装置可以为CMOS图像捕获装置。
所述短带通二向色镜120和微流控芯片210之间依次设有双带通滤波片130和双带通二向色镜140，所述第一激发光源111发出的激发光经该短带通二向色镜120透射、以及所述第二激发光源112发出的激发光经该短带通二向色镜120反射后，均入射到该双带通滤光片130，经过该双带通滤光片130后入射到双带通二向色镜140。
所述双带通二向色镜140之后的光路中，紧邻该双带通二向色镜140设有第一聚焦透镜310。
第一聚焦透镜310和第一单带通滤光片331之间设有长带通二向色镜320。第一聚焦透镜310和第二单带通滤光片332之间设有长带通二向色镜320。
在长带通二向色镜320与第一反射镜341之间设置有相应波长的第一单带通滤光片331，在长带通二向色镜320与第二反射镜342之间设置有相应波长的第二单带通滤光片332。
在第二反射镜342与图像捕获装置410之间、第三反射镜343与图像捕获装置410之间放置透镜350。
经第二反射镜342反射的荧光光束入射到透镜350。
经第一反射镜341反射的荧光光束入射到第三反射镜343、再经第三反射镜343反射后的荧光光束入射到透镜350。
入射到透镜350的光束，经透镜350汇聚于图像捕获装置410的感光面。
第一激发光源111和第二激发光源112为不同波长的LED光源，可依据实际样品需求灵活选择。两个LED光源各自出射的激发光分别直射到短带通二向色镜120，该短带通二向色镜120将两束激发光合束，该合束后的光再经双带通滤光片130直射到双带通二向色镜140，所述直射到双带通二 向色镜140的光束是双带通二向色镜140的入射光束，双带通二向色镜140将该入射光束转向后垂直入射到待测样品，之后从待测样品激发产生的荧光依次经双带通二向色镜140、第一聚焦透镜310、长带通二向色镜320、第一单带通滤光片331和第二单带通滤光片332、第一反射镜341、第二反射镜342和第三反射镜343、透镜350后，汇聚于图像捕获装置410的感光面。
具体的，本实施例的两个LED激发光源111和112的中心波长分别为470nm和625nm，两束出射的激发光直射到短带通二向色镜120。该截止波长500nm的短带通二向色镜120将两LED激发光源的出射光合束，实现双通道激发光实时同步传输，实现不同激发波长光束共用通道实时传输。
具体的，本实施例的双带通滤光片130的带通范围为461-496nm、621-653nm，该双带通滤光片130有效滤除LED光源111和112中心波长周围杂散光。
具体的，本实施例的双带通二向色镜140的带通范围为500-540nm、655-745nm，该双带通二向色镜140将激发光反射进入待测样品，样品出射的荧光透射穿过该双带通二向色镜140，以满足激发光与荧光分离，避免二者传输干扰。
该第一聚焦透镜310将透射穿过该双带通二向色镜140的出射荧光汇聚，以满足荧光光束宽度不超过长带通二向色镜320，避免丢失荧光信号，提高收集效率。
截止波长635nm的长带通二向色镜320实现不同波长荧光分通道传输,与第一反射镜341、第二反射镜342、第三反射镜343配合使用，调整出射双波长荧光光程，使得不同波长荧光到达图像捕获装置感光面时处于同一傅里叶后焦面。在本实施例中，所述样品出射的双波长荧光在图像捕获装置感光面时处于同一傅里叶后焦面。
具体的，本实施例的第一单带通滤光片331的中心波长为520nm，带宽 为40nm；第二单带通滤光片332的中心波长为690nm，带宽为50nm。二者能够有效避免出射荧光波长附近的杂散光，提高信噪比和检测灵敏度。
通过第一单带通滤光片331、第一反射镜341与第三反射镜343的短波长荧光、通过第二单带通滤光片332和第二反射镜342的长波长荧光以一定夹角α进入透镜350，其中α>0,α的值取决于第一透镜310与第二透镜350的焦距的比值。
微流控芯片210在第一聚焦透镜310前焦面上，第一聚焦透镜310后焦面与第二聚焦透镜350的前焦面重合；图像捕获装置感光面410在第一聚焦透镜310后焦面上，调整出射双波长荧光光程，使得不同波长荧光到达图像捕获装置感光面410时处于同一像面上，调节第二反射镜342和第三反射镜343倾角使不同波长荧光成像在图像捕获装置感光面410的不同位置上并且没有重合区域。
本发明的第二方面提供了一种微流控芯片实时荧光定量PCR检测的方法，采用如前所述的双通道实时荧光定量PCR仪光路系统进行检测，包括如下步骤，如图4所示：
步骤S100，配制PCR反应试剂，将待测样本加入到所述PCR反应试剂中，并将所述带有待测样本的PCR反应试剂添加到微流控芯片中。
具体的，该待测样本可以是合成的病毒质粒，按照商品化病毒核酸检测试剂盒的要求，将合成的病毒质粒，加入PCR反应试剂中，然后将带有病毒核酸质粒的PCR反应试剂，添加到微流控芯片210中。
步骤S200,将所述微流控芯片置于升降温模块上，设置所述升降温模块的温度程序进行升降温循环，并进行荧光采集。
将微流控芯片置于升降温模块上，设置升降温模块的温度程序为：例如：首先95℃加热3min，进行预变性；随后进入升降温循环：95℃加热10s进行变性，55℃加热35s进行退火延伸，共进行45个循环，实现核酸扩增。在每一次55℃加热33s的时候进行荧光采集。
步骤S300,使双色激发光的入射方向朝向微流控芯片的透光面，PCR反应试剂中的两种荧光探针上的荧光基团发出出射荧光，所述出射荧光通过双带通二向色镜，经由透镜汇聚，再通过长带通二向色镜实现不同波长荧光分通道传输,与反射镜配合使用，调整出射双波长荧光光程，成像在图像捕获装置感光面。
步骤S400,采集荧光信号，绘制荧光信号强度曲线，根据所述曲线判断目标模板的初始浓度，实现定量检测。
随着升降温循环的进行，采集到的图形的荧光信号会逐渐增强，达到一定值后保持不变，荧光强度可以绘制得到一条横坐标为时间(Ct数，循环数)，纵坐标为荧光强度的“S”型曲线，根据该曲线即可判断目标模板的初始浓度，实现新型冠状病毒核酸的定量检测。
下面以一个具体的实施例，来进一步说明选取新型冠状病毒核酸质粒作为检测的目标模板，配合商品化检测试剂盒进行检测的方法。
参照图1所示为上述光路系统配合升、降温加热模块应用于微流控芯片技术领域，实现双通道实时荧光定量PCR检测。该实施方案可以针对各种病原微生物的核酸实现特异性检测，本实施例中将具体选取新型冠状病毒核酸质粒作为检测的目标模板，配合商品化检测试剂盒进行检测。
在本实施例中，按照商品化新型冠状病毒核酸检测试剂盒的要求，配制PCR反应试剂，将合成的新型冠状病毒质粒，按照商品化试剂盒的要求，加入PCR反应试剂中。将带有新型冠状病毒核酸质粒的PCR反应试剂，添加到微流控芯片中。
将微流控芯片置于升降温模块上，设置升降温模块的温度程序为：首先95℃加热3min，进行预变性；随后进入升降温循环：95℃加热10s进行变性，55℃加热35s进行退火延伸，共进行45个循环，实现核酸扩增。在每一次55℃加热33s的时候进行荧光采集。
微流控芯片的透光面朝向双色LED激发光入射方向，试剂中的两种荧 光探针上的荧光基团发出荧光，出射荧光通过双带通二向色镜，经由透镜汇聚，再通过长带通二向色镜实现不同波长荧光分通道传输,与反射镜配合使用，调整出射双波长荧光光程，成像在图像捕获装置感光面。
随着升降温循环的进行，采集到的图形的荧光信号会逐渐增强，达到一定值后保持不变，荧光强度可以绘制得到一条横坐标为时间(Ct数，循环数)，纵坐标为荧光强度的“S”型曲线，如图3所示，根据该曲线即可判断目标模板的初始浓度，实现新型冠状病毒核酸的定量检测。
综上所述，本发明提供了一种双通道实时荧光定量PCR仪光路系统，包括双色激发模块、微流控芯片模块、双通道荧光探测模块和图像处理模块；所述双通道荧光探测模块用于对待测样品扩增，并采集荧光；所述双色激发模块用于使待测样品出射荧光，并将激发光源出射的激发光与待测样品出射的荧光分离；所述微流控芯片模块用于实现不同波长荧光分通道传输,调整出射的不同波长荧光的光程使得所述待测样品出射的不同波长荧光在所述图像处理模块的图像捕获装置的感光面处于同一傅里叶后焦面；所述图像处理模块用于感光所述荧光。本发明采用双通道激发待测样品荧光，单台图像捕获装置实现不同波长荧光实时同步成像，降低了光路系统及整体仪器的成本；光路系统设计简单、结构紧凑，减少了光路系统中的光信号传输衰减，也有利于整体仪器小型化、模块化；此光路系统也避免了激发光和荧光之间的干扰，提高了图像捕获装置接收信噪比和仪器检测灵敏度。
应当理解的是，本发明的上述具体实施方式仅仅用于示例性说明或解释本发明的原理，而不构成对本发明的限制。因此，在不偏离本发明的精神和范围的情况下所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。此外，本发明所附权利要求旨在涵盖落入所附权利要求范围和边界、或者这种范围和边界的等同形式内的全部变化和修改例。

Claims (10)

  1. 一种双通道实时荧光定量PCR仪光路系统，其特征在于，包括双色光激发模块(100)、微流控芯片模块(200)、双通道荧光探测模块(300)和图像处理模块(400)；
    所述微流控芯片模块(200)用于对待测样品扩增；
    所述双色光激发模块(100)用于使待测样品出射荧光，并将激发光源出射的激发光与待测样品出射的荧光分离；
    所述双通道荧光探测模块(300)用于实现不同波长荧光分通道传输,调整出射的不同波长荧光的光程，使得所述待测样品出射的不同波长荧光在所述图像处理模块(400)的图像捕获装置的感光面处于同一像面上并分布在感光面不同空间位置上；
    所述图像处理模块(400)用于感光所述荧光，并对图像数据进行分析。
  2. 根据权利要求1所述的光路系统，其特征在于，所述双色激发模块(100)包括两个激发光源(111，112)、短带通二向色镜(120)、双带通滤光片(130)、双带通二向色镜(140)，所述两个激发光源的激发光波长不同；
    所述微流控芯片模块(200)包括微流控芯片(210)和升降温模块(220)，待测样品夹持在所述升降温模块(220)上；
    所述双通道荧光探测模块(300)包括第一聚焦透镜(310)，长带通二向色镜(320)，单带通滤光片(331,332)，反射镜(341,342,343)和第二聚焦透镜(350)；
    所述图像处理模块(400)包括图像捕获装置(410)；
    所述短带通二向色镜(120)和微流控芯片(210)之间依次设有双带通滤波片(130)和双带通二向色镜(140)；
    紧邻所述双带通二向色镜(140)设有第一聚焦透镜(310)；
    所述聚焦透镜(310)和第一滤波片(331)之间设有长带通二向色镜(320)；
    在长带通二向色镜(320)与反射镜(341，342)之间分别设置有相应波长的单带通滤光片(331，332)；
    在第二反射镜(342)与图像捕获装置(410)之间、第三反射镜(343)与图像捕获装置(410)之间放置第二聚焦透镜(350)。
  3. 根据权利要求2所述的光路系统，其特征在于，两个所述激发光源(111，112)各自出射的激发光分别直射到短带通二向色镜(120)，所述短带通二向色镜(120)将两束激发光合束，合束后的光再经双带通滤光片(130)直射到双带通二向色镜(140)，所述直射到双带通二向色镜(140)的光束是双带通二向色镜(140)的入射光束，所述双带通二向色镜(140)将所述入射光束反射后垂直入射到待测样品，之后从待测样品激发产生的荧光依次经所述双带通二向色镜(140)、第一聚焦透镜(310)、长带通二向色镜320、单带通滤光片(331，332)、反射镜(341、342、343)、第二聚焦透镜(350)后，汇聚于图像捕获装置(410)的感光面。
  4. 根据权利要求2或3所述的光路系统，其特征在于，第一激发光源(111)发出的激发光经所述短带通二向色镜(120)透射、以及所述第二激发光源(112)发出的激发光经所述短带通二向色镜(120)反射后，均入射到所述双带通滤光片(130)，经过所述双带通滤光片(130)后入射到所述双带通二向色镜(140)。
  5. 根据权利要求2至4中任一项所述的光路系统，其特征在于，经第二反射镜(342)反射的荧光光束入射到透镜350；
    经第一反射镜(341)反射的荧光光束入射到第三反射镜(343)、再经第三反射镜(343)反射后的荧光光束入射到第二聚焦透镜(350)；
    入射到第二聚焦透镜(350)的光束，经第二聚焦透镜(350)汇聚于图像捕获装置(410)的感光面。
  6. 根据权利要求2至5中任一项所述的光路系统，其特征在于，通过第一单带通滤光片(331)、第一反射镜(341)与第三反射镜(343)的短波长荧光，以及通过第二单带通滤光片(332)、第二反射镜(342)的长波长荧光以一定夹角α进入透镜(350)。
  7. 根据权利要求6所述的光路系统，其特征在于，所述α>0,所述α的值取决于第一透镜(310)与第二透镜(350)的焦距的比值。
  8. 根据权利要求2至7中任一项所述的光路系统，其特征在于，所述微流控芯片(210)在第一聚焦透镜(310)的前焦面上，所述第一聚焦透镜(310)的后焦面与第二聚焦透镜(350)的前焦面重合，所述图像捕获装置感光面(410)在所述第一聚焦透镜(310)的后焦面上，调整出射双波长荧光光程，使得不同波长荧光到达所述图像捕获装置感光面(410)时处于同一像面上，调节所述第二反射镜(342)和所述第三反射镜(343)的倾角使不同波长荧光成像在所述图像捕获装置感光面(410)的不同位置上并且没有重合区域。
  9. 一种微流控芯片实时荧光定量PCR检测的方法，其特征在于，采用如权利要求1-8任一项所述的双通道实时荧光定量PCR仪光路系统进行检测，包括如下步骤：
    步骤S100，配制PCR反应试剂，将待测样本加入到所述PCR反应试剂中，并将所述带有待测样本的PCR反应试剂添加到微流控芯片中。
    步骤S200,将所述微流控芯片置于升降温模块上，设置所述升降温模块的温度程序进行升降温循环，并进行荧光采集。
    步骤S300,使双色激发光的入射方向朝向微流控芯片的透光面，PCR反应试剂中的两种荧光探针上的荧光基团发射荧光，所述出射荧光通过双带通二向色镜，经由透镜汇聚，再通过长带通二向色镜实现不同波长荧光分通道传输,与反射镜配合使用，调整出射双波长荧光光程，成像在图像捕获装置感光面。
    步骤S400,采集荧光信号，绘制荧光信号强度曲线，根据所述曲线判断目标模板的初始浓度，实现定量检测。
  10. 根据权利要求9所述的方法，其特征在于，所述设置所述升降温模块的温度程序进行升降温循环的步骤包括：
    第一温度加热第一时间，进行预变性；
    升降温循环：第一温度加热第二时间进行变性，第二温度加热第三时间进行退火延伸，共进行多个上述循环，实现核酸扩增；
    在每一次第二温度加热少于第三时间2秒的时候进行荧光采集。
PCT/CN2020/103159 2020-06-05 2020-07-21 一种双通道实时荧光定量pcr仪光路系统及检测方法 WO2021243823A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010503768.6A CN111589478B (zh) 2020-06-05 2020-06-05 一种双通道实时荧光定量pcr仪光路系统及检测方法
CN202010503768.6 2020-06-05

Publications (1)

Publication Number Publication Date
WO2021243823A1 true WO2021243823A1 (zh) 2021-12-09

Family

ID=72184654

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/103159 WO2021243823A1 (zh) 2020-06-05 2020-07-21 一种双通道实时荧光定量pcr仪光路系统及检测方法

Country Status (2)

Country Link
CN (1) CN111589478B (zh)
WO (1) WO2021243823A1 (zh)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114152574A (zh) * 2021-12-31 2022-03-08 天津工业大学 用于膜组件完整性检测的便携式水质分析仪及其检测方法
CN114317698A (zh) * 2021-12-17 2022-04-12 青岛松辉光电有限公司 一种反射式pcr仪
CN114414546A (zh) * 2022-01-28 2022-04-29 福州大学 一种高通量液相生物分子检测方法及装置
CN114486840A (zh) * 2022-03-03 2022-05-13 北京金竟科技有限责任公司 一种阴极荧光光谱与高衬度成像装置及其成像方法
CN115198005A (zh) * 2022-07-04 2022-10-18 北京翔东智能科技有限公司 一种基于荧光探针法检测基因突变的方法
CN115629053A (zh) * 2022-08-24 2023-01-20 北京化工大学 一种高通量蛋白质热稳定性分析仪
CN115670391A (zh) * 2023-01-03 2023-02-03 中国科学技术大学 一种用于生物组织诊断的同轴智能影像系统
CN116297378A (zh) * 2023-05-24 2023-06-23 科美诊断技术股份有限公司 光检测测量系统

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112683869B (zh) * 2020-12-25 2023-03-14 中国科学院苏州生物医学工程技术研究所 一种荧光定量检测方法
CN113122614B (zh) * 2021-04-15 2021-11-16 珠海市尚维高科生物技术有限公司 一种荧光定量pcr处理方法和系统
CN114480111A (zh) * 2022-02-15 2022-05-13 深圳阿斯克医疗有限公司 一种实时荧光定量pcr仪
CN114644980B (zh) * 2022-05-23 2022-08-30 翊新诊断技术(苏州)有限公司 一种多通道荧光pcr检测系统及多通道荧光检测方法
CN116602617A (zh) * 2022-12-30 2023-08-18 深圳市瑞沃德生命科技有限公司 光纤记录系统
CN116500010B (zh) * 2023-06-25 2024-01-26 之江实验室 荧光显微成像系统及其方法、荧光显微检测装置

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103091298A (zh) * 2013-02-01 2013-05-08 厦门大学 一种实时荧光定量pcr检测系统
CN204269552U (zh) * 2014-12-16 2015-04-15 南京融智生物科技有限公司 多色荧光检测装置
US20170370842A1 (en) * 2016-06-24 2017-12-28 Taigen Bioscience Corporation Multi-channel fluorescence detecting system and method of using the same
CN110308127A (zh) * 2019-08-15 2019-10-08 四川朴澜医疗科技有限公司 可多荧光信号同时检测的光学分析装置

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2916047B1 (fr) * 2007-05-11 2010-06-04 Force A Procede et systeme pour caracteriser un tissu biologique.
DE102007039111B4 (de) * 2007-08-18 2014-11-20 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. STED-Fluoreszenzmikroskopie mit Zweiphotonen-Anregung
JP5856814B2 (ja) * 2011-11-10 2016-02-10 オリンパス株式会社 画像解析方法および画像解析装置
US8735853B2 (en) * 2012-06-09 2014-05-27 E.I. Spectra, Llc Fluorescence flow cytometry
CN105241374B (zh) * 2015-10-16 2017-10-03 哈尔滨工程大学 双波长共路正交载频数字全息检测装置及检测方法
CN105738331B (zh) * 2016-01-29 2019-07-23 山东师范大学 一种用于单细胞电泳芯片的双激光诱导荧光多色检测器
CN107478629A (zh) * 2017-09-04 2017-12-15 中国科学院苏州生物医学工程技术研究所 一种大面积数字pcr微滴荧光高通量检测装置和方法
CN108414446A (zh) * 2018-03-30 2018-08-17 广东顺德墨赛生物科技有限公司 微流控芯片荧光检测设备、方法以及装置
US11029506B2 (en) * 2018-04-20 2021-06-08 Coluxa Inc. Scanning microscope with multiplexed light sources
CN108776122B (zh) * 2018-05-04 2022-01-04 华南师范大学 一种荧光成像方法、实时差分超分辨显微成像方法及装置
CN109030438A (zh) * 2018-07-06 2018-12-18 广州蓝勃生物科技有限公司 一种用于多波长荧光检测的光路模组
CN209117579U (zh) * 2018-09-27 2019-07-16 北京海维尔科技发展有限公司 一种新型多通道激光扫描成像仪的光学模块
CN109407295B (zh) * 2018-12-18 2020-07-24 中国科学院深圳先进技术研究院 一种基于dmd可多色激发的结构光显微系统及多色激发方法
CN110068560B (zh) * 2019-04-17 2021-08-06 深圳大学 一种受激辐射损耗超分辨成像系统及方法
CN110236694B (zh) * 2019-06-04 2021-09-10 广东欧谱曼迪科技有限公司 基于光谱响应特性的同屏近红外双谱荧光成像方法及系统

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103091298A (zh) * 2013-02-01 2013-05-08 厦门大学 一种实时荧光定量pcr检测系统
CN204269552U (zh) * 2014-12-16 2015-04-15 南京融智生物科技有限公司 多色荧光检测装置
US20170370842A1 (en) * 2016-06-24 2017-12-28 Taigen Bioscience Corporation Multi-channel fluorescence detecting system and method of using the same
CN110308127A (zh) * 2019-08-15 2019-10-08 四川朴澜医疗科技有限公司 可多荧光信号同时检测的光学分析装置

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114317698A (zh) * 2021-12-17 2022-04-12 青岛松辉光电有限公司 一种反射式pcr仪
CN114152574B (zh) * 2021-12-31 2023-10-17 天津工业大学 用于膜组件完整性检测的便携式水质分析仪及其检测方法
CN114152574A (zh) * 2021-12-31 2022-03-08 天津工业大学 用于膜组件完整性检测的便携式水质分析仪及其检测方法
CN114414546B (zh) * 2022-01-28 2023-07-28 福州大学 一种高通量液相生物分子检测方法及装置
CN114414546A (zh) * 2022-01-28 2022-04-29 福州大学 一种高通量液相生物分子检测方法及装置
CN114486840A (zh) * 2022-03-03 2022-05-13 北京金竟科技有限责任公司 一种阴极荧光光谱与高衬度成像装置及其成像方法
CN114486840B (zh) * 2022-03-03 2023-09-12 北京金竟科技有限责任公司 一种阴极荧光光谱与高衬度成像装置及其成像方法
CN115198005B (zh) * 2022-07-04 2023-09-08 北京翔东智能科技有限公司 一种基于荧光探针法检测基因突变的方法
CN115198005A (zh) * 2022-07-04 2022-10-18 北京翔东智能科技有限公司 一种基于荧光探针法检测基因突变的方法
CN115629053A (zh) * 2022-08-24 2023-01-20 北京化工大学 一种高通量蛋白质热稳定性分析仪
CN115670391A (zh) * 2023-01-03 2023-02-03 中国科学技术大学 一种用于生物组织诊断的同轴智能影像系统
CN116297378A (zh) * 2023-05-24 2023-06-23 科美诊断技术股份有限公司 光检测测量系统
CN116297378B (zh) * 2023-05-24 2023-09-15 科美诊断技术股份有限公司 光检测测量系统

Also Published As

Publication number Publication date
CN111589478B (zh) 2021-06-29
CN111589478A (zh) 2020-08-28

Similar Documents

Publication Publication Date Title
WO2021243823A1 (zh) 一种双通道实时荧光定量pcr仪光路系统及检测方法
WO2016124083A1 (zh) 一种超小型化多通道实时荧光光谱检测装置
JP2023099589A (ja) 生体サンプルを評価するためのシステムおよび方法
WO2023197734A1 (zh) 多通道超分辨基因检测仪及其检测方法
US20060134775A1 (en) Systems, illumination subsystems, and methods for increasing fluorescence emitted by a fluorophore
KR20120133654A (ko) 형광 검출 광학계 및 이를 포함하는 다채널 형광 검출 장치
CN110220872B (zh) 可携式多色荧光检测装置
JP6937697B2 (ja) 光検出システム及びその使用方法
WO2022218329A1 (zh) 一种荧光定量pcr处理方法和系统
CN104267009A (zh) 六色实时荧光定量pcr分析仪
CN112525870A (zh) 一种大面积荧光成像检测装置
WO2020000712A1 (zh) 固定波长的拉曼散射快速采集和成像设备
US8964183B2 (en) Systems and methods for screening of biological samples
KR100818351B1 (ko) 다채널 바이오 칩 스캐너
WO2003038413A1 (en) Apparatus for analyzing florescent image of biochip
EP1157268B1 (en) Imaging system for an optical scanner
JP3957118B2 (ja) 試験片およびこの試験片からの画像情報読取装置
CN211374514U (zh) 导光棒传光无透镜照明成像装置及微流控芯片检测系统
CN113189065B (zh) 光学检测方法
JP5616793B2 (ja) 検出システム及び方法
RU2304277C2 (ru) Устройство для одновременного контроля в реальном масштабе времени множества амплификаций нуклеиновой кислоты
CN220932790U (zh) 一种双光路荧光检测装置
CN117402721B (zh) 一种多色荧光检测的检测装置及检测方法
RU132203U1 (ru) Многоканальный капиллярный генетический анализатор
CN115144381B (zh) Pcr仪的发射光检测器及pcr仪

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20938705

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 27/01/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20938705

Country of ref document: EP

Kind code of ref document: A1