CN111589478B - Double-channel real-time fluorescence quantitative PCR instrument light path system and detection method - Google Patents
Double-channel real-time fluorescence quantitative PCR instrument light path system and detection method Download PDFInfo
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- CN111589478B CN111589478B CN202010503768.6A CN202010503768A CN111589478B CN 111589478 B CN111589478 B CN 111589478B CN 202010503768 A CN202010503768 A CN 202010503768A CN 111589478 B CN111589478 B CN 111589478B
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Abstract
A double-channel real-time fluorescence quantitative PCR instrument light path system and a detection method thereof are disclosed, wherein the light path system comprises a double-color light excitation module, a micro-fluidic chip module, a double-channel fluorescence detection module and an image processing module; the micro-fluidic chip module is used for amplifying a sample to be detected and collecting fluorescence; the double-color light excitation module is used for enabling the sample to be detected to emit fluorescence and separating excitation light emitted by the excitation light source from the fluorescence emitted by the sample to be detected; the double-channel fluorescence detection module is used for realizing the transmission of different wavelength fluorescence sub-channels and adjusting the optical paths of the emergent fluorescence with different wavelengths. According to the invention, two channels are adopted to excite the fluorescence of the sample to be detected, and the single image capturing device realizes real-time synchronous imaging of the fluorescence with different wavelengths, so that the cost of an optical path system and the cost of the whole instrument are reduced; the optical path system has simple design and compact structure, reduces the transmission attenuation of optical signals in the optical path system, and is beneficial to the miniaturization and modularization of the whole instrument; the interference between the excitation light and the fluorescence is avoided, and the receiving signal-to-noise ratio of the image capturing device and the detection sensitivity of the instrument are improved.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a double-channel real-time fluorescence quantitative PCR instrument optical path system and a detection method.
Background
The continuous development of life science instruments provides powerful guarantee and effective research tools for biomedicine. As a life science instrument widely applied to multiple fields of biochemical analysis, clinical diagnosis, disease control screening and the like, the PCR instrument mainly realizes the qualitative analysis of results after a large amount of specific DNA fragments are amplified in vitro in a short time according to the polymerase chain reaction technology. Early PCR instruments can only perform semi-quantitative and qualitative analysis by amplification and then by an end-point method, thus having the disadvantages of not only real-time performance but also poor detection reproducibility and certain error. Therefore, real-time fluorescence quantitative PCR instruments have come into play. The method is characterized in that fluorescent groups are combined in a PCR system, the whole PCR process is monitored in real time by utilizing fluorescent signal accumulation, and quantitative analysis is carried out through a standard curve. The technology not only realizes the qualitative and quantitative crossing of PCR, but also has the advantages of strong specificity, high efficiency, automation and the like. With the development of the fields of gene science, molecular biology and the like, people put forward more requirements on real-time fluorescent quantitative PCR instruments, most of which tend to develop towards multicolor fluorescent channels, so that multiple PCR, SNP and other analyses can be carried out simultaneously. But the requirement of the multi-channel real-time fluorescence quantitative PCR instrument is met mainly by a mode that a plurality of independent detection optical channels are parallel and share one set of detector. The method needs to select the channels and detect the channels one by one, so that the problems of long time required by overall detection, large caliber of a detector, large space required by an optical system structure, high material cost and the like are faced.
Disclosure of Invention
In order to solve the problems, the invention provides a double-channel real-time fluorescence quantitative PCR instrument optical path system and a detection method, and the optical path system combines the characteristics of microminiaturization, integration, rapid reaction, high sensitivity and the like of a microfluidic chip technology, can further widen the application field, and better promote the development of multidisciplinary advantage complementary type. The double-channel real-time fluorescence quantitative PCR instrument has compact structure of the light path system, reduces the light intensity loss of the transmission distance, realizes the real-time synchronous imaging of the fluorescence with different wavelengths, has high operation efficiency, and avoids the interference between the excitation light and the fluorescence.
The invention is realized by adopting the following technical scheme:
the invention discloses a double-channel real-time fluorescence quantitative PCR instrument optical path system, which comprises a double-color light excitation module 100, a micro-fluidic chip module 200, a double-channel fluorescence detection module 300 and an image processing module 400;
the microfluidic chip module 200 is used for amplifying a sample to be detected;
the two-color light excitation module 100 is used for enabling a sample to be detected to emit fluorescence and separating excitation light emitted by an excitation light source from the fluorescence emitted by the sample to be detected;
the dual-channel fluorescence detection module 300 is configured to implement transmission of different wavelength fluorescence in different channels, and adjust optical paths of emitted different wavelength fluorescence, so that the different wavelength fluorescence emitted from the sample to be detected is on the same image plane and distributed at different spatial positions of the light-sensitive plane on the light-sensitive plane of the image capturing device of the image processing module 400;
the image processing module 400 is configured to sense the fluorescence and analyze image data.
Further, the two-color excitation module 100 includes two excitation light sources 111, 112, a short-bandpass dichroic mirror 120, a dual-bandpass filter 130, and a dual-bandpass dichroic mirror 140, where the wavelengths of the excitation lights of the two excitation light sources are different;
the microfluidic chip module 200 comprises a microfluidic chip 210 and a temperature raising and lowering module 220, and a sample to be detected is clamped on the temperature raising and lowering module 220;
the dual-channel fluorescence detection module 300 includes a first focusing lens 310, a long bandpass dichroic mirror 320, single bandpass filters 331,332, a mirror 341,342,343, and a second focusing lens 350;
the image processing module 400 includes an image capture device 410;
a double-band-pass filter plate 130 and a double-band-pass dichroic mirror 140 are sequentially arranged between the short-band-pass dichroic mirror 120 and the microfluidic chip 210;
a first focusing lens 310 is disposed proximate to the double-bandpass dichroic mirror 140;
a long-band-pass dichroic mirror (320) is arranged between the focusing lens 310 and the first filter 331;
single band-pass filters 331,332 of corresponding wavelengths are respectively provided between the long band-pass dichroic mirror 320 and the reflecting mirrors 341, 342;
a second focusing lens 350 is disposed between the second mirror 342 and the image capturing apparatus 410, and between the third mirror 343 and the image capturing apparatus 410.
Further, the excitation light emitted from each of the two excitation light sources 111, 112 is directly emitted to the short-band-pass dichroic mirror 120, the short-band-pass dichroic mirror 120 combines the two excitation light beams, the combined light is directly emitted to the double-band-pass dichroic mirror 140 through the double-band-pass filter 130, the light beam directly emitted to the double-band-pass dichroic mirror 140 is an incident light beam of the double-band-pass dichroic mirror 140, the incident light beam is reflected by the double-band-pass dichroic mirror 140 and then perpendicularly emitted to the sample to be measured, and then the fluorescence generated by excitation of the sample to be measured sequentially passes through the double-band-pass dichroic mirror 140, the first focusing lens 310, the long-band-pass dichroic mirror 320, the single-band-pass filters 331,332, the reflectors 341,342,343, and the second focusing lens 350 and then is converged on the photosensitive surface of.
Further, 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, and then both enters the dual bandpass filter 130, and enters the dual bandpass dichroic mirror 140 after passing through the dual bandpass filter 130.
Further, the fluorescent light beam reflected by the second mirror 342 is incident on the lens 350;
the fluorescent light beam reflected by the first reflecting mirror 341 is incident to the third reflecting mirror 343, and the fluorescent light beam reflected by the third reflecting mirror 343 is incident to 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 by the second focusing lens 350.
Further, 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 enter the lens 350 at an included angle α.
Further, α >0, the value of α depends on the ratio of the focal lengths of the first lens 310 and the second lens 350.
Further, the microfluidic chip 210 is located 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, the light-sensing surface 410 of the image capturing device is located on the back focal plane of the first focusing lens 310, the optical path of the outgoing dual-wavelength fluorescence is adjusted, so that the different-wavelength fluorescence reaches the light-sensing surface 410 on the same image plane, and the tilt angles of the second reflecting mirror 342 and the third reflecting mirror 343 are adjusted, so that the different-wavelength fluorescence is imaged on different positions of the light-sensing surface 410 without coinciding areas.
The second aspect of the invention discloses a method for real-time fluorescence quantitative PCR detection of a microfluidic chip, which adopts the optical path system of the dual-channel real-time fluorescence quantitative PCR instrument to carry out detection and comprises the following steps:
step S100, preparing a PCR reaction reagent, adding a sample to be detected into the PCR reaction reagent, and adding the PCR reaction reagent with the sample to be detected into a microfluidic chip;
step S200, placing the microfluidic chip on a temperature raising and lowering module, setting a temperature program of the temperature raising and lowering module to carry out temperature raising and lowering circulation, and carrying out fluorescence collection;
step S300, enabling the incident direction of the bicolor exciting light to face the light-transmitting surface of the microfluidic chip, enabling fluorescent groups on two fluorescent probes in the PCR reaction reagent to emit fluorescent light, enabling the emergent fluorescent light to pass through a dual-band-pass dichroic mirror and be converged through a lens, enabling the different-wavelength fluorescent light to be transmitted in different channels through the long-band-pass dichroic mirror, enabling the different-wavelength fluorescent light to be matched with a reflector for use, adjusting the optical path of the emergent dual-wavelength fluorescent light, and imaging the light on the light-sensing surface of the;
and S400, collecting fluorescence signals, drawing a fluorescence signal intensity curve, and judging the initial concentration of the target template according to the curve to realize quantitative detection.
Further, the step of setting the temperature program of the temperature raising and lowering module to perform temperature raising and lowering circulation includes:
heating at a first temperature for a first time to perform pre-denaturation;
heating and cooling circulation: heating at the first temperature for a second time for denaturation, heating at the second temperature for a third time for annealing extension, and performing a plurality of cycles in total to realize nucleic acid amplification;
fluorescence collection was performed at each second temperature heating for less than 2 seconds of the third time.
In summary, the optical path system comprises a bicolor excitation module, a microfluidic chip module, a two-channel fluorescence detection module and an image processing module; the double-channel fluorescence detection module is used for amplifying a sample to be detected and collecting fluorescence; the double-color excitation module is used for enabling the sample to be detected to emit fluorescence and separating excitation light emitted by the excitation light source from the fluorescence emitted by the sample to be detected; the micro-fluidic chip module is used for realizing the transmission of different wavelength fluorescence sub-channels, and adjusting the optical paths of the emitted different wavelength fluorescence to ensure that the different wavelength fluorescence emitted by the sample to be detected is positioned at different spatial positions of the light-sensitive surface of the image capturing device of the image processing module; the image processing module is used for sensing the fluorescence.
According to the invention, the fluorescence of the sample to be detected is excited by adopting two channels, and the single image capturing device realizes real-time synchronous imaging of the fluorescence with different wavelengths, so that the cost of an optical path system and the cost of the whole instrument are reduced; the optical path system has simple design and compact structure, reduces the transmission attenuation of optical signals in the optical path system, and is also beneficial to the miniaturization and modularization of the whole instrument; the optical path system also avoids the interference between the excitation light and the fluorescence, and improves the receiving signal-to-noise ratio of the image capturing device and the detection sensitivity of the instrument.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the invention adopts two channels to excite the fluorescence of the sample to be detected, and the single image capturing device realizes the real-time synchronous imaging of the fluorescence with different wavelengths, thereby reducing the cost of an optical path system and the whole instrument;
(2) the double-channel real-time fluorescence quantitative PCR instrument optical path system has a compact structure, reduces the light intensity loss of transmission distance, avoids the interference between excitation light and fluorescence, and improves the receiving signal-to-noise ratio of an image capturing device and the detection sensitivity of the instrument;
(3) the detection method realizes real-time synchronous imaging of the fluorescence with different wavelengths, has high running efficiency, and avoids the interference between the excitation light and the fluorescence;
(4) the optical path system is simple in design and compact in structure, reduces the transmission attenuation of optical signals in the optical path system, and is also beneficial to miniaturization and modularization of the whole instrument;
(5) the optical path system provided by the invention combines the characteristics of miniaturization, integration, rapid reaction, high sensitivity and the like of the microfluidic chip technology, can further widen the application field, and better promotes the development of multidisciplinary advantage complementary type.
Drawings
FIG. 1 is a schematic diagram of the optical path system of the two-channel real-time fluorescence quantitative PCR instrument of the present invention;
FIG. 2 is a diagram of the optical path system of the two-channel real-time fluorescence quantitative PCR instrument of the present invention;
FIG. 3 is a graph showing the amplification curve of the real-time fluorescent quantitative PCR detection of the microfluidic chip according to the present invention;
FIG. 4 is a flow chart of the method for real-time fluorescence quantitative PCR detection of the microfluidic chip of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings in conjunction with the following detailed description. It should be understood that the description is intended to be exemplary only, and is not intended to limit the scope of the present invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Technical term interpretation:
the invention provides a double-channel real-time fluorescence quantitative PCR instrument optical path system, which can be applied to the real-time fluorescence quantitative PCR detection of a microfluidic chip and can support the detection of emergent fluorescence with at least two wavelengths. As shown in fig. 1, the optical path system includes a two-color excitation module 100, a microfluidic chip module 200, a two-channel fluorescence detection module 300, and an image processing module 400. Specifically, 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 raising and lowering module 220, and a sample to be tested is clamped on the temperature raising and lowering module 220.
The dual-channel fluorescence detection module 300 includes a first focusing lens 310, a long bandpass dichroic mirror 320, first and second single bandpass filters 331 and 332, first, second, and third mirrors 341 and 342, and a second focusing lens 350.
The image processing module 400 includes an image capture device 410, which may be a CMOS image capture device.
A double-bandpass filter 130 and a double-bandpass dichroic mirror 140 are sequentially arranged between the short-bandpass dichroic mirror 120 and the microfluidic chip 210, and excitation light emitted by the first excitation light source 111 is transmitted through the short-bandpass dichroic mirror 120, and excitation light emitted by the second excitation light source 112 is reflected by the short-bandpass dichroic mirror 120, then is incident on the double-bandpass filter 130, and then is incident on the double-bandpass dichroic mirror 140 after passing through the double-bandpass filter 130.
A first focusing lens 310 is disposed adjacent to the double-bandpass dichroic mirror 140 in the optical path after the double-bandpass dichroic mirror 140.
A long bandpass dichroic mirror 320 is provided between the first focusing lens 310 and the first single bandpass filter 331. A long bandpass dichroic mirror 320 is disposed between the first focusing lens 310 and the second single bandpass filter 332.
A first single bandpass filter 331 of a corresponding wavelength is disposed between the long bandpass dichroic mirror 320 and the first reflecting mirror 341, and a second single bandpass filter 332 of a corresponding wavelength is disposed between the long bandpass dichroic mirror 320 and the second reflecting mirror 342.
The fluorescent light beam reflected by the second mirror 342 is incident on the lens 350.
The fluorescent light beam reflected by the first reflecting mirror 341 is incident on the third reflecting mirror 343, and the fluorescent light beam reflected by the third reflecting mirror 343 is incident on the lens 350.
The light beam incident on the lens 350 is converged 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 with different wavelengths, and can be flexibly selected according to actual sample requirements. Excitation light emitted by the two LED light sources respectively penetrates the short-band-pass dichroic mirror 120 directly, the short-band-pass dichroic mirror 120 enables the two beams of excitation light to be combined into a beam, the combined light penetrates the double-band-pass dichroic mirror 140 through the double-band-pass filter 130 directly, the beam penetrating the double-band-pass dichroic mirror 140 directly is an incident beam of the double-band-pass dichroic mirror 140, the incident beam is deflected by the double-band-pass dichroic mirror 140 and then perpendicularly enters a sample to be detected, and then fluorescence generated by excitation of the sample to be detected sequentially passes through the double-band-pass dichroic mirror 140, the first focusing lens 310, the long-band-pass dichroic mirror 320, the first single-band-pass filter 331, the second single-band-pass filter 332, the first reflecting mirror 341, the second reflecting mirror 342, the third reflecting mirror 343 and the lens 350 and then converges on a photosensitive surface of.
Specifically, the center wavelengths of the two LED excitation light sources 111 and 112 of the present embodiment are 470nm and 625nm, respectively, and the two emitted excitation lights are directly incident on the short-bandpass dichroic mirror 120. The short-band-pass dichroic mirror 120 with the cut-off wavelength of 500nm combines emergent light of two LED excitation light sources, so that real-time synchronous transmission of dual-channel excitation light is realized, and real-time transmission of light beams with different excitation wavelengths sharing channels is realized.
Specifically, the bandpass ranges of the dual bandpass filter 130 of the embodiment are 461-.
Specifically, the bandpass range of the dual-bandpass dichroic mirror 140 of the present embodiment is 500-.
The first focusing lens 310 converges the emitted fluorescence transmitted through the dual-bandpass dichroic mirror 140 to satisfy the requirement that the width of the fluorescence beam does not exceed the long-bandpass dichroic mirror 320, so as to avoid losing the fluorescence signal and improve the collection efficiency.
The long-band-pass dichroic mirror 320 with the cut-off wavelength of 635nm realizes the transmission of different wavelength fluorescent light sub-channels, and is used in cooperation with the first reflector 341, the second reflector 342 and the third reflector 343 to adjust the optical path of the emergent dual-wavelength fluorescent light, so that the different wavelength fluorescent light is positioned on the same Fourier back focal plane when reaching the light sensing surface of the image capturing device. In this embodiment, the two-wavelength fluorescence emitted by the sample is at the same fourier back focal plane when the image capture device is exposed to light.
Specifically, the center wavelength of the first single bandpass filter 331 of the present embodiment is 520nm, and the bandwidth is 40 nm; the second single bandpass filter 332 has a center wavelength of 690nm and a bandwidth of 50 nm. The two can effectively avoid stray light near the emergent fluorescence wavelength, and improve the signal-to-noise ratio and the detection sensitivity.
The short wavelength fluorescence passing through the first single band pass 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 enter the lens 350 at an included angle α, where α >0, and the value of α depends on the ratio of the focal lengths of the first lens 310 and the second lens 350.
The microfluidic chip 210 is arranged on the front focal plane of the first focusing lens 310, and the back focal plane of the first focusing lens 310 is superposed with the front focal plane of the second focusing lens 350; the image capturing device photosensitive surface 410 is on the back focal plane of the first focusing lens 310, the optical path of the outgoing dual-wavelength fluorescence is adjusted so that the fluorescence with different wavelengths reaches the image capturing device photosensitive surface 410 and is on the same image plane, and the inclination angles of the second mirror 342 and the third mirror 343 are adjusted so that the fluorescence with different wavelengths is imaged on different positions of the image capturing device photosensitive surface 410 and has no overlapping area.
The second aspect of the present invention provides a method for real-time fluorescence quantitative PCR detection of a microfluidic chip, which uses the optical path system of the dual-channel real-time fluorescence quantitative PCR instrument for detection, and includes the following steps, as shown in fig. 4:
and S100, preparing a PCR reaction reagent, adding a sample to be detected into the PCR reaction reagent, and adding the PCR reaction reagent with the sample to be detected into the microfluidic chip.
Specifically, the sample to be detected can be a synthesized virus plasmid, the synthesized virus plasmid is added into a PCR reaction reagent according to the requirement of a commercial virus nucleic acid detection kit, and then the PCR reaction reagent with the virus nucleic acid plasmid is added into the microfluidic chip.
And S200, placing the microfluidic chip on a temperature raising and lowering module, setting a temperature program of the temperature raising and lowering module to carry out temperature raising and lowering circulation, and carrying out fluorescence acquisition.
The micro-fluidic chip is arranged on the temperature rising and lowering module, and the temperature procedure of the temperature rising and lowering module is as follows: for example: firstly, heating at 95 ℃ for 3 min for carrying out pre-denaturation; and then entering a temperature rise and reduction cycle: heating at 95 ℃ for 10s for denaturation, heating at 55 ℃ for 35s for annealing extension, and performing 45 cycles in total to realize nucleic acid amplification. Fluorescence collection was performed at each heating time of 33s at 55 ℃.
And step S300, enabling the incident direction of the bicolor exciting light to face the light-transmitting surface of the microfluidic chip, enabling fluorescent groups on two fluorescent probes in the PCR reaction reagent to emit emergent fluorescent light, enabling the emergent fluorescent light to pass through the dual-band-pass dichroic mirror and be converged by the lens, enabling the different-wavelength fluorescent light to be transmitted by the long-band-pass dichroic mirror through the sub-channels, matching with the reflector for use, adjusting the optical path of the emergent dual-wavelength fluorescent light, and imaging on the light-sensing surface of the image capturing device.
And S400, collecting fluorescence signals, drawing a fluorescence signal intensity curve, and judging the initial concentration of the target template according to the curve to realize quantitative detection.
Along with the temperature rise and decrease circulation, the fluorescence signal of the collected graph can be gradually enhanced and is kept unchanged after reaching a certain value, the fluorescence intensity can be drawn to obtain an S-shaped curve with the abscissa as time (Ct number and cycle number) and the ordinate as the fluorescence intensity, the initial concentration of the target template can be judged according to the curve, and the quantitative detection of the novel coronavirus nucleic acid is realized.
The following is a specific example to further illustrate the method of selecting a novel coronavirus nucleic acid plasmid as a target template for detection, and detecting the coronavirus nucleic acid plasmid by matching with a commercial detection kit.
Referring to fig. 1, the optical path system is matched with a heating module for raising and lowering temperature, and is applied to the technical field of microfluidic chips to realize double-channel real-time fluorescent quantitative PCR detection. The embodiment can realize specific detection aiming at nucleic acid of various pathogenic microorganisms, and in the embodiment, a novel coronavirus nucleic acid plasmid is specifically selected as a target template for detection and matched with a commercial detection kit for detection.
In this example, PCR reagents were prepared according to the requirements of a commercial coronavirus nucleic acid detection kit, and the synthesized novel coronavirus plasmid was added to the PCR reagents according to the requirements of the commercial kit. Adding the PCR reaction reagent with the novel coronavirus nucleic acid plasmid into a microfluidic chip.
The micro-fluidic chip is arranged on the temperature rising and lowering module, and the temperature procedure of the temperature rising and lowering module is as follows: firstly, heating at 95 ℃ for 3 min for carrying out pre-denaturation; and then entering a temperature rise and reduction cycle: heating at 95 ℃ for 10s for denaturation, heating at 55 ℃ for 35s for annealing extension, and performing 45 cycles in total to realize nucleic acid amplification. Fluorescence collection was performed at each heating time of 33s at 55 ℃.
The light transmitting surface of the micro-fluidic chip faces the incident direction of the excitation light of the bicolor LED, the fluorescent groups on the two fluorescent probes in the reagent emit fluorescence, the emergent fluorescence passes through the dual-band-pass dichroic mirror and is converged by the lens, the transmission of different wavelength fluorescence sub-channels is realized by the long-band-pass dichroic mirror, the dual-wavelength fluorescence optical path is adjusted by matching with the reflecting mirror, and the image is imaged on the light sensing surface of the image capturing device.
Along with the increase and decrease of the temperature cycle, the fluorescence signal of the collected graph can be gradually enhanced and is kept unchanged after reaching a certain value, the fluorescence intensity can be drawn to obtain an S-shaped curve with the abscissa as time (Ct number and cycle number) and the ordinate as the fluorescence intensity, as shown in figure 3, the initial concentration of the target template can be judged according to the curve, and the quantitative detection of the novel coronavirus nucleic acid is realized.
In summary, the invention provides a two-channel real-time fluorescence quantitative PCR instrument optical path system, which comprises a two-color excitation module, a micro-fluidic chip module, a two-channel fluorescence detection module and an image processing module; the double-channel fluorescence detection module is used for amplifying a sample to be detected and collecting fluorescence; the double-color excitation module is used for enabling the sample to be detected to emit fluorescence and separating excitation light emitted by the excitation light source from the fluorescence emitted by the sample to be detected; the micro-fluidic chip module is used for realizing the transmission of different wavelength fluorescence sub-channels, and adjusting the optical paths of the emitted different wavelength fluorescence to ensure that the different wavelength fluorescence emitted by the sample to be detected is positioned on the same Fourier back focal plane on the light-sensitive surface of the image capturing device of the image processing module; the image processing module is used for sensing the fluorescence. According to the invention, the fluorescence of the sample to be detected is excited by adopting two channels, and the single image capturing device realizes real-time synchronous imaging of the fluorescence with different wavelengths, so that the cost of an optical path system and the cost of the whole instrument are reduced; the optical path system has simple design and compact structure, reduces the transmission attenuation of optical signals in the optical path system, and is also beneficial to the miniaturization and modularization of the whole instrument; the optical path system also avoids the interference between the excitation light and the fluorescence, and improves the receiving signal-to-noise ratio of the image capturing device and the detection sensitivity of the instrument.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explaining the principles of the invention and are not to be construed as limiting the invention. Therefore, any modification, equivalent replacement, improvement and the like made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. Further, it is intended that the appended claims cover all such variations and modifications as fall within the scope and boundaries of the appended claims or the equivalents of such scope and boundaries.
Claims (8)
1. A double-channel real-time fluorescence quantitative PCR instrument optical path system is characterized by comprising a double-color light excitation module (100), a micro-fluidic chip module (200), a double-channel fluorescence detection module (300) and an image processing module (400);
the microfluidic chip module (200) is used for amplifying a sample to be detected;
the double-color light excitation module (100) is used for enabling a sample to be detected to emit fluorescence and separating excitation light emitted by the excitation light source from the fluorescence emitted by the sample to be detected;
the dual-channel fluorescence detection module (300) is used for realizing the transmission of different wavelength fluorescence sub-channels and adjusting the optical paths of the emitted different wavelength fluorescence, so that the different wavelength fluorescence emitted by the sample to be detected is positioned on the same image plane on the light-sensitive surface of the image capturing device of the image processing module (400) and is distributed on different spatial positions of the light-sensitive surface;
the image processing module (400) is used for sensing the fluorescence and analyzing image data;
the two-color excitation module (100) comprises 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), wherein the wavelengths of the excitation light of the two excitation light sources are different;
the microfluidic chip module (200) comprises a microfluidic chip (210) and a temperature rising and lowering module (220), and a sample to be detected is clamped on the temperature rising and lowering module (220);
the dual-channel fluorescence detection module (300) comprises a first focusing lens (310), a long band-pass dichroic mirror (320), single band-pass filters (331,332), a reflector (341,342,343) and a second focusing lens (350);
the image processing module (400) comprises an image capturing device (410);
a double-band-pass filter plate (130) and a double-band-pass dichroic mirror (140) are sequentially arranged between the short-band-pass dichroic mirror (120) and the microfluidic chip (210);
a first focusing lens (310) is disposed proximate to the dual-bandpass dichroic mirror (140);
a long-band-pass dichroic mirror (320) is arranged between the focusing lens (310) and the first filter plate (331);
single band-pass filters (331,332) with corresponding wavelengths are respectively arranged between the long band-pass dichroic mirror (320) and the reflecting mirrors (341, 342);
placing a second focusing lens (350) between the second mirror (342) and the image capture device (410), and between the third mirror (343) and the image capture device (410);
excitation light emitted by the two excitation light sources (111, 112) respectively directly irradiates to a short-band-pass dichroic mirror (120), the short-band-pass dichroic mirror (120) combines the two beams of excited light, the combined light is directly irradiated to the double-band-pass dichroic mirror (140) through the double-band-pass filter (130), the light beam directed to the dichroic mirror (140) is an incident light beam of the dichroic mirror (140), the incident light beam is reflected by the double-bandpass dichroic mirror (140) and then vertically enters a sample to be detected, and then fluorescence generated by excitation of the sample to be detected sequentially passes through the double-bandpass dichroic mirror (140), the first focusing lens (310), the long-bandpass dichroic mirror (320), the single-bandpass filters (331,332), the reflectors (341,342,343) and the second focusing lens (350) and then is converged on a photosensitive surface of an image capturing device (410).
2. The optical path system according to claim 1, wherein the excitation light emitted from the first excitation light source (111) is transmitted through the short-bandpass dichroic mirror (120), and the excitation light emitted from the second excitation light source (112) is reflected by the short-bandpass dichroic mirror (120), and then both enters the dual-bandpass filter (130), passes through the dual-bandpass filter (130), and enters the dual-bandpass dichroic mirror (140).
3. The optical path system according to claim 2, wherein the fluorescent light beam reflected by the second mirror (342) is incident on the lens (350);
the fluorescent light beam reflected by the first reflecting mirror (341) is incident to a third reflecting mirror (343), and the fluorescent light beam reflected by the third reflecting mirror (343) is incident to a second focusing lens (350);
the light beam incident on the second focusing lens (350) is converged on the photosensitive surface of the image capturing device (410) through the second focusing lens (350).
4. The optical path system of claim 3, wherein the short wavelength fluorescence passing through the first single band pass 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), the second mirror (342), enter the lens (350) at an angle α.
5. The optical path system according to claim 4, characterized in that α >0, the value of α depending on the ratio of the focal lengths of the first lens (310) and the second lens (350).
6. The optical path system of claim 5, wherein the microfluidic chip (210) is on a front focal plane of a first focusing lens (310), a back focal plane of the first focusing lens (310) is coincident with a front focal plane of a second focusing lens (350), the image capture device light sensing plane (410) is on the back focal plane of the first focusing lens (310), the emergent dual-wavelength fluorescence optical path length is adjusted so that different-wavelength fluorescence reaches the image capture device light sensing plane (410) on the same image plane, and the inclination angles of the second mirror (342) and the third mirror (343) are adjusted so that different-wavelength fluorescence is imaged on different positions of the image capture device light sensing plane (410) without a coincident region.
7. A method for real-time fluorescence quantitative PCR detection of a microfluidic chip is characterized in that the detection is carried out by adopting the optical path system of the double-channel real-time fluorescence quantitative PCR instrument as claimed in any one of claims 1 to 6, and comprises the following steps:
step S100, preparing a PCR reaction reagent, adding a sample to be detected into the PCR reaction reagent, and adding the PCR reaction reagent with the sample to be detected into a microfluidic chip;
step S200, placing the microfluidic chip on a temperature raising and lowering module, setting a temperature program of the temperature raising and lowering module to carry out temperature raising and lowering circulation, and carrying out fluorescence collection;
step S300, enabling the incident direction of the bicolor exciting light to face the light-transmitting surface of the microfluidic chip, enabling fluorescent groups on two fluorescent probes in the PCR reaction reagent to emit fluorescent light, enabling the emergent fluorescent light to pass through a dual-band-pass dichroic mirror and be converged through a lens, enabling the different-wavelength fluorescent light to be transmitted in different channels through the long-band-pass dichroic mirror, enabling the different-wavelength fluorescent light to be matched with a reflector for use, adjusting the optical path of the emergent dual-wavelength fluorescent light, and imaging the light on the light-sensing surface of the;
and S400, collecting fluorescence signals, drawing a fluorescence signal intensity curve, and judging the initial concentration of the target template according to the curve to realize quantitative detection.
8. The method of claim 7, wherein the step of setting the temperature program of the warming and cooling module to perform a warming and cooling cycle comprises:
heating at a first temperature for a first time to perform pre-denaturation;
heating and cooling circulation: heating at the first temperature for a second time for denaturation, heating at the second temperature for a third time for annealing extension, and performing a plurality of cycles in total to realize nucleic acid amplification;
fluorescence collection was performed at each second temperature heating for less than 2 seconds of the third time.
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