WO2022141674A1 - 一种pcr检测仪及其方法 - Google Patents

一种pcr检测仪及其方法 Download PDF

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Publication number
WO2022141674A1
WO2022141674A1 PCT/CN2021/071820 CN2021071820W WO2022141674A1 WO 2022141674 A1 WO2022141674 A1 WO 2022141674A1 CN 2021071820 W CN2021071820 W CN 2021071820W WO 2022141674 A1 WO2022141674 A1 WO 2022141674A1
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Prior art keywords
excitation light
light
sample
order difference
light source
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PCT/CN2021/071820
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English (en)
French (fr)
Inventor
任鲁风
蒋鹏翀
范东雨
蔡亦梅
高静
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北京中科生仪科技有限公司
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Application filed by 北京中科生仪科技有限公司 filed Critical 北京中科生仪科技有限公司
Priority to EP21912457.5A priority Critical patent/EP4273547A1/en
Priority to JP2022552708A priority patent/JP7438580B2/ja
Publication of WO2022141674A1 publication Critical patent/WO2022141674A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6484—Optical fibres
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention requires the priority of the Chinese patent application filed on December 30, 2020 with the application number 202011643279.7 and the invention name is "a PCR detector and method thereof", and also claims the same as on December 30, 2020.
  • the invention relates to the field of PCR detection, in particular to a PCR detection instrument and a method thereof.
  • Real-time fluorescence quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve.
  • Ct value In fluorescence quantitative PCR technology, there is a very important concept - Ct value.
  • C stands for Cycle
  • t stands for threshold
  • the meaning of Ct value is: the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value.
  • Patent document CN107923922A discloses an improved subassembly and control method for use in a diagnostic assay system adapted to receive an assay cartridge.
  • Such sub-assemblies include: optical detection/excitation means.
  • the excitation block 910 includes an LED light source 911 that guides light through a filter and a lens 912 and then a rod lens 913 to emit light of a desired wavelength to a desired location of the reaction vessel 33 .
  • Optical detection block 920 includes photodiode detector 921 that detects light emitted from reaction vessel 33, which passes through rod lens 923 and filters and lenses before being received by photodiode detector 921 922 to ensure detection of a specific wavelength that may indicate a reaction corresponding to the presence of the target analyte within the reaction vessel 33.
  • the optical excitation component 910 and the optical detection component 920 are positioned on an optical mount adapted to receive the planar reaction vessel 33 .
  • the optical excitation component 910 is positioned to emit excitation energy through the edge (minor surface) of the flat surface of the reaction vessel 33, and the optical detection component 920 is positioned along the main flat surface of the reaction vessel.
  • the optical actuation component and the optical detection component are orthogonal to each other.
  • the principle of fluorescence picking is a conventional algorithm (filters filter bandpass).
  • the patent document CN111157497A discloses an excitation light source for a handheld detector, which includes: a PCB board, a light-emitting lamp group, an optical filter, an optical fiber coupler, an optical fiber, an optical fiber combiner, and an optical fiber collimator , housing, and bottom plate; wherein, the multiple groups of the light-emitting lamp groups arranged in parallel emit light of different wavelengths after being denoised by the filter, and then enter the corresponding optical fiber through the optical fiber coupler, and in the optical fiber After mixing in the beam combiner, the target is finally fluorescently excited by the optical fiber collimator; the light-emitting lamp group has a filter.
  • both the excitation light source and the reflected light source need to be used for filtering, and the photodiode detector is used for detection, and the filtered emission light is used for the detection.
  • the detection of the detector only one kind of emitted light can be detected, and a variety of different emitted lights cannot be detected at the same time.
  • the distribution of the fluorescent detection substance in the reaction chamber may be uneven or even unable to be distributed in the reaction chamber.
  • the present invention provides a PCR detector and a method thereof, wherein no filter is used after excitation light and emission light, and a spectrometer is used to detect the emission light, which can detect simultaneously. Emitted light of different wavelengths.
  • the present invention provides a PCR detector, comprising an excitation light source module, a chip device and a detection part; an excitation light source module, a chip device and a detection part, the excitation light source module emits excitation light of a preset wavelength to the chip device, when the amplification reaction is performed, the detection part is arranged on the side of the reaction chamber of the chip device;
  • the chip device includes a reaction chamber, and the reaction chamber can accommodate a sample to be detected; wherein, the sample to be detected is a nucleic acid fragment solution containing fluorescent labels;
  • the excitation light source module is used for emitting excitation light, and the excitation light emitted by the excitation light source module can irradiate the detected sample placed in the reaction chamber;
  • the excitation light source module can emit excitation light of at least two different frequency bands
  • the reaction chamber is arranged in the excitation light emission direction of the excitation light module, the detection part is arranged on one side of the reaction chamber, and the emission light formed after the excitation light irradiates the reaction chamber can be detected by the detection part ; wherein, the emission direction of the excitation light is located below the detection part, and the detection part is used to detect the emission light in the vertical direction emitted by the detected sample due to the irradiation of the excitation light;
  • the detection part includes a spectrometer; the wavelength range of the detection spectrum of the spectrometer is 340-850 nm; the spectrometer can detect the excitation light and the emission light.
  • the spectrometer detects the frequency band and intensity of excitation light in at least two different frequency bands, and the spectrometer detects the excitation light in at least two different frequency bands and the frequency band and intensity of the emission light caused by the excitation light irradiating the detection sample.
  • the excitation light frequency bands of the at least two different frequency bands include at least a first frequency band of excitation light and a second frequency band of excitation light
  • the frequency bands of emission light caused by the excitation light of the two different frequency bands include at least a first frequency band of emission light and a second frequency band of emission light.
  • the second frequency band of light; the first frequency band of excitation light, the second frequency band of excitation light, the first frequency band of emission light and the frequency band of the second frequency band of emission light have no overlapping frequency range.
  • Frequency range there is no overlapping frequency range between the excitation light frequency bands of the excitation light of at least two different frequency bands, so there is no overlapping between the different emission light frequency bands of the emission light caused by the excitation light of the at least two different frequency bands.
  • Frequency range there is no overlapping frequency range between each excitation light frequency band and each emission light frequency band caused by excitation light.
  • the excitation light source module includes a plurality of light emitting units, and the light emitting units are light emitting diodes or semiconductor lasers.
  • the excitation light source module includes a PCB board, an optical fiber combiner, at least one light-emitting diode, and at least one optical fiber corresponding to each light-emitting diode;
  • the light emitting diodes are arranged on one side of the PCB board; an optical fiber coupler is arranged at the output end of each light emitting diode, and each optical fiber coupler is respectively coupled with an optical fiber, and the excitation light of the corresponding wavelength is coupled into the optical fiber through the optical fiber coupler, and transmission through optical fibers;
  • the optical fiber combiner arranges and bundles each optical fiber in a preset manner, and an optical fiber collimator is also arranged at the output end of the optical fiber combiner to convert the excitation light in the optical fiber into collimated light;
  • the at least one light emitting diode is used for emitting excitation light of at least two different frequency bands, and the frequency ranges of the excitation light of the at least two different frequency bands do not have overlapping frequency ranges.
  • the chip device includes a sample application layer and a pipeline layer, the sample application layer and the pipeline layer are arranged in sequence from top to bottom, the sample application layer includes a sample addition hole and a reagent tube, and the sample addition hole
  • the reagent tube is used to transport the buffer solution
  • the pipeline layer includes a reaction chamber, the reaction chamber is pre-embedded with a freeze-dried reagent, and the freeze-dried reagent contains a fluorescent labeling substance, wherein the sample addition hole The sample inside is mixed with the buffer solution and then enters the reaction chamber to obtain the tested sample.
  • the fluorescently labeled substances are at least two of fam, hex, cy5, and cy5.5, wherein the detected samples fluorescently labeled with cy5 and cy5.5 cannot be detected simultaneously, and the fluorescently labeled samples of fam and hex are detected Samples also cannot be tested simultaneously.
  • the excitation light source module further includes a light source rotating device, the light source rotating device is used to rotate the PCB board provided with a plurality of light emitting units, that is, the light source rotating device can be set on the PCB board of the present invention, so that due to excitation The light spot of the emitted light caused by the light is evenly distributed in the reaction chamber, which makes the detection by the detection part more accurate.
  • a light source rotating device is used to rotate the PCB board provided with a plurality of light emitting units, that is, the light source rotating device can be set on the PCB board of the present invention, so that due to excitation The light spot of the emitted light caused by the light is evenly distributed in the reaction chamber, which makes the detection by the detection part more accurate.
  • the light emitting unit is an LD light source or an LED light source.
  • the fluorescent labels of the sample to be detected are fam, hex, cy5cy5.5, and when it is an LD light source, the fluorescently labeled detected samples of fam and cy5.5 can be detected simultaneously.
  • the invention provides a kind of PCR detection method, comprising:
  • a spectrometer was used to collect cycle 0-40 fluorescence intensity A i , A i , i ⁇ [0,40].
  • the collected cycle 0-40 fluorescence intensity A i normalization processing method specifically includes:
  • first-order difference, second-order difference, and third-order difference processing on the D i obtained after normalization which are respectively:
  • it also includes taking the maximum value of the second-order difference, specifically:
  • polynomial fitting is also included, and the above 3 clicks are also included.
  • it also includes calculating the Ct value
  • negative/positive discrimination is also included:
  • the detector provided by the present invention adopts a spectrometer and does not use a filter, which simplifies the structure of the structure and reduces the number of parts;
  • the spectrometer provided by the present invention can detect multiple different spectra at the same time, thereby realizing the simultaneous detection of multiple fluorescent labels, that is, multiple different nucleic acids can be detected simultaneously; the detection efficiency is improved, and the use of filters can only detect one at a time.
  • the emission light is limited, and the direction of the emission light provided by the present invention is perpendicular to the direction of the detection part, so as to avoid the phenomenon of excessive explosion caused by the excitation light being too strong.
  • the detection method provided by the present invention ensures that the method is simple, the accuracy rate is high, and the reliability of the discrimination between negative and positive is high.
  • the excitation light module provided by the present invention is provided with a light source rotating device, and the light source rotating device drives the light emitting unit on the PCB board to rotate, so that the light spot distribution of the emitted light is more uniform.
  • Fig. 1 is the fluorescence spectrum that the detection part of the present invention detects and obtains;
  • Fig. 2 is a fluorescence value-emission peak comparison diagram before and after single-channel fam amplification provided by the present invention
  • Fig. 3 is the schematic diagram of fam channel fluorescence value and temperature cycle provided by the present invention.
  • Fig. 4 is the curve that the Fam channel provided by the present invention takes the emission peak fluorescence value by cycle
  • FIG. 5 is a structural diagram of a light source-chip-spectrometer provided by the present invention.
  • FIG. 8 is a schematic structural diagram of a chip device provided by the present invention.
  • FIG. 9 is a schematic structural diagram of the overall sample loading layer of the excitation light source module provided by the present invention.
  • FIG. 10 is a schematic structural diagram of the pipeline layer of the overall sample loading layer of the excitation light source module according to the embodiment of the present invention.
  • Fig. 11 is the Ct value that the embodiment of the present invention obtains.
  • Figure 12 is the distribution of the fluorescent labeling substance provided by the present invention on the emission light spot
  • FIG. 13 is the distribution of the fluorescent labeling substance on the emission light spot after adjustment by the light source rotating device provided by the present invention.
  • the invention provides a PCR detector, comprising an excitation light source module, a chip and a detection part; an excitation light source module, a chip device and a detection part, the excitation light source module emits excitation light of a preset wavelength to the chip device, and the process is carried out During the amplification reaction, the detection part is arranged above the reaction chamber of the chip device;
  • the chip device includes a reaction chamber, and the reaction chamber can accommodate a sample to be detected; wherein, the sample to be detected is a nucleic acid fragment solution containing fluorescent labels;
  • the excitation light source module is used for emitting excitation light, and the excitation light emitted by the excitation light source module can irradiate the detected sample placed in the reaction chamber;
  • the excitation light source module can emit excitation light of at least two different frequency bands
  • the reaction chamber is arranged in the excitation light emission direction of the excitation light module, the detection part is arranged on one side of the reaction chamber, and the emission light formed after the excitation light irradiates the reaction chamber can be detected by the detection part ;
  • the emission direction of the excitation light is the horizontal direction
  • the emission direction of the excitation light is vertical to the detection part
  • the detection part is used to detect the emission light in the vertical direction emitted by the sample to be detected due to the irradiation of the excitation light ;
  • the detection part provided by the present invention includes a spectrometer; the wavelength range of the detection spectrum of the spectrometer is 340-850 nm; the spectrometer detects excitation light and emission light.
  • the spectrometer detects frequency bands and intensities of excitation light in at least 2 different frequency bands, and the spectrometer detects the frequency bands and intensities of excitation light and induced emission light in at least 2 different frequency bands.
  • the present invention is a schematic diagram of the results of simultaneous detection of two channels of fluorescent labeling substances Fam and cy5.5 by a spectrometer; wherein, EX1 in Figure 1 is the peak of fam excitation light, EM1 is the peak of fam emission light, EX1 is the peak value of the excitation light of cy5.5, and EM1 is the peak value of the emission light of cy5.5;
  • the excitation light of two different frequency bands includes the first frequency band of excitation light and the second frequency band of excitation light.
  • the frequency bands of the second frequency band, the first frequency band of the emitted light, and the second frequency band of the emitted light do not overlap or partially overlap each other.
  • the spectrometer can detect fam excitation light, fam emission light, cy5.5 excitation light and cy5.5 emission light at the same time, and the fluorescent labeling substance can be fam, hex, cy5, cy5.5, etc., as shown in the following table.
  • the LD light source When it is an LD light source, it can simultaneously detect the samples labeled with fam and cy5.5, but the samples labeled with cy5 and cy5.5 cannot be detected at the same time, and the samples labeled with fam and hex can also be detected. cannot be detected simultaneously.
  • the excitation wavelength of the fam fluorescent label is 494nm
  • the emission wavelength is 518nm
  • the excitation wavelength of the cy5.5 fluorescent label is 675nm
  • the emission wavelength is 695nm, that is, the fam fluorescence
  • the excitation wavelength and emission wavelength of the label and the excitation wavelength and emission wavelength of the cy5.5 fluorescent label have no completely overlapping frequency bands.
  • the range of the first frequency band of the excitation light is 450-480nm
  • the range of the second frequency band of the excitation light is 540-560nm, that is, the range of the first frequency band of the excitation light and the range of the second frequency band of the excitation light are completely non-overlapping frequency bands. scope.
  • the first frequency band range of the excitation light is 520-550 nm
  • the second frequency band range of the excitation light is 540-560 nm, that is, the first frequency band range of the excitation light and the second frequency band range of the excitation light are partially non-overlapping frequency ranges.
  • the excitation light source provided by the present invention can be a list of the wavelength of the LD light source, the excitation wavelength and the emission wavelength of the fluorescent labeling substances fam, hex, cy5, and cy5.5, as shown in Table 1 below.
  • Table 1 is a list of the wavelengths of the LD light source, the excitation wavelengths and the emission wavelengths of the fluorescent labeling substances fam, hex, cy5, and cy5.5;
  • the spectrometer detects the frequency band and intensity of excitation light of at least two different frequency bands, and the spectrometer detects the excitation light of at least two different frequency bands and the frequency band and intensity of the emission light caused by the excitation light irradiating the detection sample.
  • the excitation light frequency bands of the at least two different frequency bands include at least the first frequency band of the excitation light and the second frequency band of the excitation light, and the frequency bands of the emission light caused by the excitation light of the two different frequency bands include at least the first frequency band of the emission light and the second frequency band of the emission light.
  • Frequency band; the first frequency band of excitation light, the second frequency band of excitation light, the first frequency band of emission light and the frequency band of the second frequency band of emission light have no overlapping frequency range.
  • the chip device provided by the present invention includes a sample application layer and a pipeline layer, the sample application layer and the pipeline layer are arranged in sequence from top to bottom, and the sample application layer includes sample application holes and reagent tubes , the sample addition hole is used to add samples, the reagent tube is used to transport the buffer solution, the pipeline layer includes a reaction chamber, the reaction chamber is pre-embedded with a freeze-dried reagent, and the freeze-dried reagent contains a fluorescent labeling substance , wherein the sample in the sample addition hole is mixed with the buffer solution and then enters the reaction chamber to obtain the detected sample.
  • the buffer solution is a buffer solution
  • the buffer solution refers to a mixed solution composed of weak acids and their salts, weak bases and their salts, which can offset and reduce the influence of external strong acids or strong bases on the pH of the solution to a certain extent.
  • the pH of the solution is relatively stable.
  • the excitation light source module provided by the present invention further includes a light source rotating device, the light source rotating device is used to rotate the PCB board provided with a plurality of light emitting units, that is, the light source rotating device can be set on the PCB board of the present invention , so that the light spot of the emitted light caused by the excitation light is evenly distributed in the reaction chamber, so that the detection part can detect more accurately.
  • FIG. 5 is a schematic diagram of a light source-chip-spectroscope optical circuit diagram according to an embodiment of the present invention
  • the system of this embodiment includes a chip device 30 , an excitation light module 6 , a temperature control module 4 and a detection part 5 , and the excitation light module 6.
  • the fluorescence detection unit 5 obtains fluorescence information to complete the detection.
  • the temperature control module 4 controls the temperature in the chip device, so that the The reagents reach the best reaction state.
  • the detection part 5 is arranged above the reaction chamber of the chip device.
  • the apparatus to which the emitted light detection method provided by the embodiment of the present invention is applied includes: an excitation light source module 6 , a sample to be detected 21 , and a detection unit 5 , and a light emission unit illuminates the sample to be detected 21 to excite the sample to be detected 21 .
  • the sample to be detected 21 contains a fluorescent group to generate emission light under the excitation of the excitation light;
  • the The detection part is arranged on the optical path of the emitted light to capture and detect the emitted light; specifically, an optical path diagram is shown in FIG. 6 , and different arrows are used for the optical path of the excitation light and the optical path of the emitted light Represented to show the difference between the two lights.
  • the function of the detection part 6 is to capture and detect the emitted light.
  • the spectrometer may be a fluorescence spectrometer, which allows simultaneous reading of multiple emission lights and excitation lights, so that it can be test.
  • the spectrometer provided in the present invention does not include a filter, therefore, excitation light of multiple different frequency bands and emission light caused by excitation light can be detected simultaneously.
  • the spectrometer can be C12889MA from Hamamatsu.
  • the excitation light source module includes a plurality of light emitting units, and the light emitting units are light emitting diodes or semiconductor lasers.
  • the excitation light source module includes a PCB board, a fiber combiner, at least one light-emitting diode, and at least one optical fiber corresponding to each light-emitting diode;
  • the light emitting diodes are arranged on one side of the PCB board; an optical fiber coupler is arranged at the output end of each light emitting diode, and each optical fiber coupler is respectively coupled with an optical fiber, and the excitation light of the corresponding wavelength is coupled into the optical fiber through the optical fiber coupler, and transmission through optical fibers;
  • the optical fiber combiner arranges and bundles each optical fiber in a preset manner, and an optical fiber collimator is also arranged at the output end of the optical fiber combiner to convert the excitation light in the optical fiber into collimated light;
  • the at least one light emitting diode is used for emitting excitation light of at least two different frequency bands, and the frequency ranges of the excitation light of the at least two different frequency bands do not have overlapping frequency ranges.
  • the difference between the present invention and the excitation light source module in the prior art is that the excitation light source module provided by the present invention does not include the filter 63 , while the excitation light source module in the prior art must include a filter 63;
  • the optical excitation component provided by the present invention includes an excitation light source module, and the excitation light module 6 adopts a light-emitting module with a smaller volume, and switches the excitation light source in the excitation source module by using a switch to generate different excitation light sources. wavelength of excitation light.
  • the excitation light module 6 includes a PCB board 61 for carrying the excitation light source, such as an LED light source or an LD light source, or other light sources.
  • the LED light source is arranged on one side of the PCB board.
  • the LED light source includes several arranged LEDs
  • the lamp 62 is provided with an optical fiber coupler 64 at the front end of each LED lamp, each optical fiber coupler 64 is respectively coupled with an optical fiber 65, and the excitation light of the corresponding wavelength is coupled into the optical fiber through the optical fiber coupler 64, and through the optical fiber transmission; also includes a fiber combiner 66, which arranges and bundles each optical fiber in a preset manner, and an optical fiber collimator 67 is also provided at the output end of the optical fiber combiner 66 to combine the The excitation light is converted into collimated excitation light.
  • the excitation light source module provided by the present invention further includes a light source rotating device, and the light source rotating device is used to rotate the PCB board provided with a plurality of light emitting units, that is, the light source rotating device can rotate the PCB board, Therefore, the light emitting unit, ie, the LED light source or the LD light source, is driven to rotate, that is, a light source rotating device can be provided on the PCB board of the present invention, so that the light spot of the emitted light caused by the excitation light is evenly distributed in the reaction chamber, so that the detection part can detect more accurately.
  • the light source rotating device is used to rotate the PCB board provided with a plurality of light emitting units, that is, the light source rotating device can rotate the PCB board, Therefore, the light emitting unit, ie, the LED light source or the LD light source, is driven to rotate, that is, a light source rotating device can be provided on the PCB board of the present invention, so that the light spot of the emitted light caused by the excitation
  • Figure 12 shows the distribution of the fluorescent labeling substances on the reaction chamber 112 when the excitation light source module provided by the present invention is not rotated, that is, is not adjusted, that is, the distribution is uneven, It may not even be distributed on the reaction chamber, or the fluorescent labeling substance is not distributed on the reaction chamber; as shown in FIG. 12, FIG. 13 is the distribution of the fluorescent labeling substance on the reaction chamber 112 after adjustment by the light source rotating device provided by the present invention. In some cases, due to the adjustment of the light source rotating device, the distribution of fluorescent labeling substances in the reaction chamber 112 is uniform and can be distributed in the same reaction chamber, solving the technical problem of uneven distribution in the prior art, as shown in FIG. 13 .
  • the working process of the excitation light source module start the LED lamp 62, emit the LED light source, and introduce the excitation light source of the corresponding wavelength into the optical fiber through the optical fiber coupler, and the multiple optical fibers are converged through the optical fiber combiner 66, and the excitation light source transmitted therein is combined into a One beam, and finally convert the transmitted light into collimated excitation light through a fiber collimator and irradiate it to the target for detection; there are multiple LED lights, which can emit excitation light of different wavelengths at the same time, and no filter is required.
  • the invention uses a spectrometer to identify different wavelengths of emitted light, so no filters are needed.
  • the reactor provided by the present invention is a chip device.
  • the chip device of the present invention includes a sample application layer 3 arranged at the uppermost end, a gasket 2 arranged on the lower side of the sample application layer 3, and a The pipeline layer 101 on the lower side of the gasket 2 and the sealing film 104 arranged on the lowermost side, wherein the sample adding hole 302 is provided on the upper side of the sample adding layer 3 for adding the sample into the chip and injecting the chip into the chip.
  • the sample inside is extracted, purified, amplified and reacted.
  • the sample application layer and the pipeline layer in this embodiment are movably connected to the limit frame 106 disposed on the side of the pipeline layer 101 through the clips 304 , and correspondingly, a first card slot is provided on the inner side of the limit frame 106 107.
  • the first card slots are connected to each other through the card strips, so as to realize the switching and fixing of the relative positions of the sample application layer and the pipeline layer.
  • the sealing film is pasted on the lower side of the pipeline layer 101 to achieve sealing.
  • a second clamping slot 109 is also provided on the side of the limiting frame on the lower side of the first clamping slot 107.
  • the side wall of the gasket 2 is also provided with a second clamping strip (not shown in the figure).
  • the sliding connection between the gasket 2 and the pipeline layer 101 is realized by the mutual cooperation of the second clamping strip and the second clamping groove, and the relative positions of the two can be switched and fixed.
  • the lower side of the gasket 2 in the embodiment of the present invention is further provided with a first sliding rail 202.
  • a second sliding groove 108 is provided on the upper side of the pipeline layer 101. The first sliding rail 202 passes through the second sliding groove.
  • the second chute 108 in this embodiment is disposed on the inner side of the limiting frame 106 on the pipeline layer.
  • the end of the gasket 2 is provided with a plurality of notches and protrusions arranged alternately, wherein the first slide rail 202 is provided on the bottom surface of the outermost protrusion.
  • the sample introduction hole of the present invention is provided with a sample introduction hole cover 303 for sealing.
  • the sample application layer and the pipeline layer are also provided with a buckle structure, and a first buckle 301 is arranged on one side of the sample application layer, and the protruding end of the lower side of the first buckle 301 protrudes from the bottom end of the sample application layer , after the sample adding layer and the pipeline layer are installed together, the first clip is clamped on the side of the pipeline layer to prevent the sample adding layer and the pipeline layer from being separated.
  • two first single valves 102 are arranged on the pipeline layer for injecting samples and reaction reagents into the pipeline respectively; double valves 103 are also arranged on the pipeline layer for injecting reagents and samples,
  • the double valve 103 is communicated with the reaction chamber 112 through a pipeline, and as shown in FIG. 5 , handles 201 are also provided on both sides of the gasket 2 to facilitate the extraction of the chip device.
  • the reaction chamber is arranged on the edge of the pipeline layer, and the reaction chamber has a semi-elliptical structure, which can not only make the reaction reagents react, but also can pass the protruding semi-elliptical shape during use. The structure realizes convenient positioning and installation.
  • a row of puncture needles 105 is arranged on the pipeline layer of the present invention.
  • the puncture needle is connected to the reagent in the sample application layer, and the fluorescent sequence marked in the reagent is generated with the nucleic acid puncture needle at the corresponding position.
  • a set of probe sequences whose sequences are completely complementary can be obtained by determining the position of the probe with the strongest fluorescence intensity.
  • a baffle plate is also arranged on the outer side of the puncture needle, which plays a blocking and positioning role when the sample application layer is matched with the pipeline layer.
  • the sample adding layer 3 in the sample adding state, several groups of reagent tubes are arranged in the sample adding layer, and the sample adding layer 3 is locked with the first card slot 107 by the clips 304 on it.
  • the sample adding layer 3 cooperates with the pipeline layer 101 from top to bottom, and the puncture needle is isolated from the reagent in the reagent tube by the gasket 2, so as to prevent the puncture needle and the reagent from being mixed due to vibration during transportation. puncture.
  • the gasket 2 is pulled out along the second chute 108.
  • the sample application layer 3 is pressed down, so that the clips 304 on the sample application layer are pressed down. It is clamped with the second clamping slot 109.
  • the puncture needle disposed on the pipeline layer is mixed with the reagent in the sample application layer, and the reagent is introduced into the pipeline layer for measurement.
  • the chip device can be well preserved during the storage of reagents and transportation, and the reagents can be introduced into the pipeline layer by simply pulling out the gasket during use.
  • FIG. 9 is a schematic structural diagram of the sample adding layer according to the embodiment of the present invention.
  • the sample adding chamber is located below the sample adding hole 302 in this embodiment, and the sample adding chamber can be connected to a reagent tube for loading reagents.
  • the lower part of the sample chamber is provided with a reagent outlet 312, and a sealing structure is arranged between the reagent outlet 312 and the sample addition chamber for sealing.
  • a pressurizing structure is also provided on one side of the sample adding chamber, which includes a tube wall 305, and a piston 308 is arranged inside the tube wall.
  • the piston 308 moves toward the sample adding chamber, and pushes the reagent in it to flow out to the reagent outlet;
  • the end of the piston rod of the piston 308 is provided with a sealing ring 311 for sealing.
  • the piston rod of this embodiment is also provided with a nut 307, which is threadedly connected to the nut 307 to achieve relative rotational motion.
  • an output structure such as a cylinder, an oil cylinder, is provided at one end of the piston rod.
  • the piston rod can also be connected by a rotating output structure, such as a motor and a lead screw. At this time, the piston rod performs a rotary motion, and only needs to be able to push the reagent to flow out to the reagent outlet.
  • a guide sleeve 306 is set on the outer side of the nut, and a corresponding shaft shoulder is set on the inner side of the pipe wall to position and fix the guide sleeve 306; , used to clamp the corresponding guide sleeve 306 .
  • a sheath 309 is also provided on the outer side of the guide sleeve 306 to protect the piston rod, the nut, and the guide sleeve.
  • the efficiency of reagent injection can also be achieved by the outward suction of other reagent tubes and the inward push of the current actual tube to achieve efficient reagent injection.
  • several groups of reagent tubes are arranged.
  • five groups of reagent tubes are arranged to apply reagents to the pipeline layer.
  • the reagents can be lysis solution, eluent or cleaning solution, etc., which can greatly improve the use efficiency.
  • a second buckle 310 is provided below the sample application layer, and the second buckle is arranged on the side opposite to the first buckle to prevent the sample application layer from sliding.
  • the pipeline layer in this embodiment is provided with the reaction chamber 112 , the first buffer chamber 110 , the second buffer chamber 111 , and the sample The purified purification bin 114, wherein the first end of the double valve is connected to the purification bin 114 through the first pipeline 118 and the second pipeline 115; the second end of the double valve is communicated with the second buffer bin 111, and the second buffer bin 111 is connected to the first buffer silo 110 through a pipeline, and a pipeline branch is arranged on the pipeline between the first buffer silo and the second buffer silo, a second single valve is arranged on the pipeline branch, and the pipeline branch is another One end is connected to the purification tank 114, and the first buffer tank is connected to the purification tank 114 through the third pipeline 116 and the fourth pipeline 117; a number of connection holes 119 are also provided on the pipeline layer for connection.
  • the first pipeline 118 includes a vertical pipeline and a horizontal pipeline, and the eluted nucleic acid material enters the amplification chamber through long-distance transportation
  • the second pipeline 115 includes a vertical pipeline and a horizontal pipeline , one end of which is connected with the purification chamber, and the other end is connected with the first pipeline as a whole.
  • the third pipeline 116 is a multi-directional bending tube, one end of which is connected to the first buffer tank, and the other end is connected to the first liquid inlet; one end of the fourth pipeline 117 is connected to the first buffer tank. One liquid inlet is connected, the other end is connected with the purification chamber, and the fourth pipeline 117 is also provided with a sample inlet and a first single valve.
  • the sample inlet is used to add the sample
  • the first liquid inlet is used to add the lysis solution
  • the first single valve is opened, so that the sample and the lysis solution are mixed and reacted.
  • the connected piston rod and the piston rod connected to the first liquid inlet perform a push and suction operation to achieve full mixing of the sample and the lysate to generate the first reactant, which is a liquid
  • the liquid passes through the fourth pipeline 117
  • the purification chamber has built-in magnetic beads
  • the purification chamber is a reaction chamber for nucleic acid extraction and purification, then close the first single valve, open the second single valve, and inject the second single valve into the second liquid inlet.
  • the second reagent is a cleaning solution
  • the second reagent enters the purification chamber through the pipeline connected to the second reagent port, cleans the substances in the purification chamber, and injects the third reagent into the third liquid inlet
  • the third reagent is a cleaning solution
  • the third reagent enters the purification chamber through the pipeline connected to it, cleans the nucleic acid substance in the purification chamber again, and injects the fourth reagent into the fourth liquid inlet.
  • the fourth reagent is an eluent, the fourth reagent enters the purification chamber through the pipeline connected to it, and the nucleic acid substance in the purification chamber is eluted from the magnetic beads set thereon to obtain nucleic acid substance, and the nucleic acid substance in the purification chamber is eluted.
  • the nucleic acid substance is introduced into the reaction chamber 112 through the first pipeline 115 and the second pipeline 118 to perform an amplification reaction.
  • the piston moves to the sample adding chamber to increase the pressure in it, so as to push the reagent to flow to the reagent outlet to realize the injection of the reagent or sample;
  • Applying samples or reagents to the pipeline layer can greatly improve the efficiency of use.
  • the amplification chamber is arranged on the edge of the pipeline layer, and the reaction chamber 112 is a semi-elliptical structure, which can not only make the reaction reagents react, but also can realize convenient positioning and operation through the protruding semi-elliptical structure during use. mounted, where the chip device is transparent.
  • the invention provides a gasket and a related connection structure, on the one hand, the sample layer and the pipeline layer can be connected well, avoiding vibration, and on the other hand, the needle can have a better placement space, the gasket and The sample layer is slidingly installed for easy disassembly.
  • the sample adding layer cooperates with the pipeline layer from top to bottom, and the puncture needle is isolated from the reagent in the reagent tube by the gasket to prevent the puncture needle from mixing with the reagent due to vibration during transportation and avoid puncture.
  • pull the gasket out along the second chute When the test needs to be carried out, pull the gasket out along the second chute. After the gasket is pulled out along the second chute, press down the sample layer so that the clip on the sample layer is in contact with the second clip.
  • the puncture needle arranged on the pipeline layer is mixed with the reagent in the sample application layer, and the reagent is introduced into the optical path layer for measurement.
  • the chip device can be well preserved during storage of reagents and during transportation, and when in use, the reagents can be introduced into the pipeline layer by simply pulling out the gasket.
  • Figure 2 provided by the present invention is a comparison diagram of fluorescence value-emission peak before and after single-channel fam amplification; wherein, Cycle0 is the fluorescence spectrum curve that is not amplified, and cycle 45 is the fluorescence spectrum curve after 45 cycles of amplification Fluorescence spectrum curve;
  • Figure 3 provided by the present invention is a schematic diagram of fam channel fluorescence value and temperature cycle; wherein the abscissa is the time, and the ordinate is the fluorescence value at the emission peak of the fam channel, from which it can be seen that the fluorescence
  • the change trend of the curve with the temperature F5 is the change curve of the fluorescence value with time
  • F6 is the change curve of the temperature with time
  • Figure 3 leads to the curve of the fluorescence value according to the cycle in Figure 4
  • Figure 4 is the Fam channel provided by the present invention according to the cycle Take the curve of the emission peak fluorescence value, the time point of lighting is 3s before the end of the extension stage
  • a PCR detection method includes:
  • a 1 to A 40 are used as signals, and the base A 0 is subtracted

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Abstract

本发明提供一种PCR检测仪，属于PCR检测技术领域，包括激发光源模块、芯片装置和检测部；激发光源模块、芯片装置和检测部，所述反应仓设置于所述激发光模块的激发光发射方向，所述检测部设置于反应仓的一侧，所述激发光照射所述反应仓后形成的发射光能够被检测部检测；其中，其激发光的发射方向位于检测部的下方，所述检测部用于检测被检测样品因受激发光的照射发出的在垂直方向上的发射光；所述检测部包括光谱仪；所述光谱仪检测光谱的波长范围是340-850nm；所述光谱仪检测激发光和发射光。光谱仪可以同时检测多路不同光谱，从而实现同时检测多个荧光标记，即可以同时检测多种不同核酸。

Description

一种PCR检测仪及其方法
本发明要求于2020年12月30日提交中国专利局，申请号为202011643279.7、发明名称为“一种PCR检测仪及其方法”的中国专利申请的优先权，同时要求与2020年12月30日提交中国专利局，申请号为202023334358.8、发明名称为“一种PCR检测仪”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明涉及PCR检测领域，具体涉及一种PCR检测仪及其方法。
背景技术
实时荧光定量PCR技术，是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。
在荧光定量PCR技术中，有一个很重要的概念-Ct值。C代表Cycle，t代表threshold，Ct值的含义是：每个反应管内的荧光信号到达设定的域值时所经历的循环数。
专利文献CN107923922A公开了用于在适于接收化验盒的诊断化验系统中使用的改进的子组件和控制方法。这种子组件包括：光学检测/激励装置。其中，激励块910包括LED光源911，LED光源911引导光通过滤光片和透镜912，然后通过棒状透镜913，从而将期望波长的光发射到反应容器33的期望位置。光学检测块920包括光电二极管检测器921，该光电二极管检测器921检测从反应容器33发射的光，在由光电二极管检测器921接收之前，发射的光穿过棒状透镜923以及滤光片和透镜922，以确保检测特定波长，特定波长可指示对应于反应容器33内目标分析物的存在的反应。同时光学激励部件910和光学检测部件920定位在适于接收平面反应容器33的光学安装件上。光学激励部件910定位成通过反应容器33的平坦表面的边缘(副面)发射激励能量，并且光学检测部件920沿着反应容器的主平坦表面定位。在一个方面，光学激励部件和光学检测部件彼此正交。其荧光采点原理为常规算法(滤光片过滤带通)。如图7所示，专利文献CN111157497A公开了一种手持式检测仪用激发光源，其包括：PCB板、发光灯组、滤光片、光纤耦合器、光纤、光纤合束器、光纤准直器、外壳、底板；其中，并联设置的多组所述发光灯组发出不同波长的光通过所述滤光片去噪后，经过所述光纤耦合器进入对应所述光纤中，并在所述光纤合束器中混合，最终通过所述光纤准直器对目标进行荧光激发；其发光灯组是具有滤光片的。
总之，现有技术中为了检测目标分析物，均需要使用激发光源和反射光源后均会使用滤光组件进行滤光处理，而且使用光电二极管检测器进行检测，而使用滤光处理后的发射光进入检 测器检测，只能检测一种发射光，而不能同时检测多种不同的发射光。另外，在检测时，荧光检测物质分配在反应仓可能会存在不均匀甚至不能在反应仓中分布的现象。
发明内容
为解决现有技术中存在的上述问题，本发明提供了一种PCR检测仪及其方法，其激发光和发射光后均不使用滤光片，并采用光谱仪对发射光进行检测，能够同时检测不同波长的发射光。
本发明提供的具体技术方案如下：
一方面，本发明提供了一种PCR检测仪，包括激发光源模块、芯片装置和检测部；激发光源模块、芯片装置和检测部，所述激发光源模块发出预设波长的激发光至所述芯片装置，在进行扩增反应时，所述检测部设置在所述芯片装置的反应仓一侧；
所述芯片装置包括反应仓，反应仓内能够容纳被检测样品；其中，被检测样品为含有荧光标记的核酸片段溶液；
所述激发光源模块用于发射激发光，激发光源模块发射的激发光能够照射置于反应仓的所述被检测样品；
所述激发光源模块能够发射至少2个不同频段的激发光；
其中，所述反应仓设置于所述激发光模块的激发光发射方向，所述检测部设置于反应仓的一侧，所述激发光照射所述反应仓后形成的发射光能够被检测部检测；其中，激发光的发射方向位于检测部的下方，所述检测部用于检测被检测样品因受激发光的照射发出的在垂直方向上的发射光；
所述检测部包括光谱仪；所述光谱仪检测光谱的波长范围是340-850nm；所述光谱仪能够检测到所述激发光和所述发射光。
优选地，所述光谱仪检测至少2个不同频段的激发光的频段和强度，所述光谱仪检测至少2个不同频段的激发光和由激发光照射检测样品引起的发射光的频段和强度。
优选地，所述至少2个不同频段的激发光频段至少包括激发光第一频段和激发光第二频段，2个不同频段的激发光引起的发射光的频段至少包括发射光第一频段和发射光第二频段；所述激发光第一频段、激发光第二频段、发射光第一频段和发射光第二频段的频段两两之间均无相互重合的频段范围。
优选地，至少2个不同频段的激发光的激发光频段之间无相互重合的频段范围，所至少2个不同频段的激发光引起的所述发射光的不同发射光频段之间无相互重合的频段范围；各个激发光频段以及因激发光引起的各个发射光频段之间无相互重合的频段范围。
优选地，所述激发光源模块包括多个光发射单元，所述光发射单元为发光二极管或半导体激光器。
优选地，所述激发光源模块包括PCB板、光纤合束器、至少一个发光二极管以及至少一根与每一个发光二极管对应的光纤；
发光二极管设置于PCB板一侧；在每一发光二极管的输出端设置有一光纤耦合器，每一光纤耦合器分别与一光纤耦合，通过光纤耦合器将对应波长的激发光耦合于光纤中，并通过光纤传输；
光纤合束器将各个光纤按预设方式排列合束为一体，在所述的光纤合束器的输出端还设置有光纤准直器，用以将光纤内的激发光转变成准直光；
所述至少一个发光二极管用于发射至少2个不同频段的激发光，所述至少2个不同频段的激发光的频率范围无相互重合的频率范围。
优选地，所述芯片装置包括加样层和管路层，所述加样层、管路层从上至下依次设置，所述加样层包括加样孔和试剂管，所述加样孔用于添加样品，所述试剂管用于输送buffer溶液，所述管路层包括反应仓，所述反应仓内预埋有冻干试剂，所述冻干试剂包含荧光标记物质，其中，加样孔内的样品和buffer溶液混合后进入反应仓，从而得到被检测样品。
优选地，所述的荧光标记的物质为fam、hex、cy5、cy5.5的至少两种，其中cy5、cy5.5荧光标记的被检测样品不能被同时检测，fam、hex荧光标记的被检测样品也不能被同时检测。
优选地，所述激发光源模块的还包括光源旋转装置，所述光源旋转装置用于使设置有多个光发射单元PCB板旋转，即本发明的PCB板上可设置光源旋转装置，使得因激发光引起的发射光的光斑在反应仓的分布均匀，使得检测部检测更加准确。
优选地，光发射单元为LD光源或LED光源。
优选地，被检测样品的荧光标记为fam,hex,cy5cy5.5,其中，当为LD光源时，可同时检测fam和cy5.5的荧光标记的被检测样品。
另一方面，本发明提供了一种PCR检测方法，包括：
采用光谱仪采集cycle 0-40荧光强度A i，A i,i∈[0,40]。
优选地，还包括对采集的cycle 0-40荧光强度A i作归一化处理，得到D i＝C i-y i,i∈[1,40]。
优选地，采集的cycle 0-40荧光强度A i归一化处理方法具体包括：
扣除基底：A 1～A 40作为信号，减去基底A 0；
B i＝A i-A 0,i∈[1,40]
平滑数据：对数据进行平滑处理；
Figure PCTCN2021071820-appb-000001
确定基线：选择C 3～C 10，根据最小二乘法拟合出直线作为基线
yi＝ax i+b,i∈[0,40]
数据扣除baseline，得到D i＝C i-y i,i∈[1,40]。
优选地，还包括对归一化处理后得到D i的作一阶差分、二阶差分和三阶差分处理，分别为：
一阶差分
Figure PCTCN2021071820-appb-000002
二阶差分
Figure PCTCN2021071820-appb-000003
三阶差分
D i″′＝D″ i+1-D i″,i∈]4,36[。
优选地，还包括取二阶差分最大值，具体为：
选出三阶差分中符合“正负负负”→“正负负”→“正负”规律的最大点，且要求对应cycle一阶差分大于0，该坐标+1即为二阶差分最大值对应坐标，另±1共计3点。
优选地，还包括多项式拟合，将上述3点按
二次函数(y＝ax 2+bx+c)拟合，取
Figure PCTCN2021071820-appb-000004
优选地，还包括求Ct值，
过X处做D i拟合曲线的切线，其与baseline交点的横坐标即为Ct值，进而可得threshold，
Figure PCTCN2021071820-appb-000005
优选地，还包括阴/阳性判别：
若Ct值≥38，判断为阴性；反之，则为阳性。
优选地，还包括绘图，
将D i按cycle数绘制曲线。
与现有技术相对比，本发明的有益效果如下：
(1)本发明提供的检测仪采用光谱仪，不使用滤镜，简化了结构的构成，减少了零部件；
(2)本发明提供的光谱仪可以同时检测多路不同光谱，从而实现同时检测多个荧光标记，即可以同时检测多种不同核酸；提高了检测效率，打破了使用滤光镜一次只能检测一个发射光的局限，而且本发明提供的发射光的方向与检测部的方向垂直，避免激发光太强而出现过爆的现象。
(3)本发明提供的检测方法，如求Ct值的方法保证了其方法简单，准确率高，而且使得阴性和阳性的判别的可信度高。
(4)本发明提供的激发光模块设置有光源旋转装置，光源旋转装置带动PCB板上的光发射单元旋转，使得发射光的光斑分配的更加均匀。
附图说明
图1为本发明检测部检测得到的荧光光谱；
图2为本发明提供的单通道fam扩增前后荧光值-发射峰对比图；
图3为本发明提供的fam通道荧光值与温度循环示意图；
图4为本发明提供的Fam通道按cycle取发射峰荧光值的曲线；
图5为本发明提供的光源-芯片-光谱仪结构图；
图6为本发明提供的光源-芯片-光谱仪光路图；
图7为现有技术提供的激发光源模块结构示意图；
图8为本发明提供的芯片装置的结构示意图；
图9为本发明提供的的激发光源模块整体加样层的结构示意图；
图10为本发明实施例的激发光源模块整体加样层的管路层的结构示意图；
图11为本发明实施例得到的Ct值；
图12为本发明提供的荧光标记物质在发射光光斑上的分布情况；
图13为本发明提供的光源旋转装置调整后荧光标记物质在发射光光斑上的分布情况。
具体实施方式
下面结合附图，对本发明提供的一种PCR检测仪及其方法具体说明。
本发明提供了一种PCR检测仪，包括激发光源模块、芯片和检测部；激发光源模块、芯片装置和检测部，所述激发光源模块发出预设波长的激发光至所述芯片装置，在进行扩增反应时，所述检测部设置在所述芯片装置的反应仓上方；
所述芯片装置包括反应仓，反应仓内能够容纳被检测样品；其中，被检测样品为为含有荧光标记的核酸片段溶液；
所述激发光源模块用于发射激发光，激发光源模块发射的激发光能够照射置于反应仓的所述被检测样品；
所述激发光源模块能够发射至少2个不同频段的激发光；
其中，所述反应仓设置于所述激发光模块的激发光发射方向，所述检测部设置于反应仓的一侧，所述激发光照射所述反应仓后形成的发射光能够被检测部检测；其中，激发光的发射方向为水平方向，其激发光的发射方向与检测部是垂直的，所述检测部用于检测被检测样品因受激发光的照射发出的在垂直方向上的发射光；
其中，本发明提供的所述检测部包括光谱仪；所述光谱仪检测光谱的波长范围是340-850nm；所述光谱仪检测激发光和发射光。
所述光谱仪检测至少2个不同频段的激发光的频段和强度，所述光谱仪检测至少2个不同频段的激发光和引起的发射光的频段和强度。
如图1所示，本发明通过光谱仪对荧光标记物质Fam和cy5.5两个通道同时检测的结果示意图；其中，图1中的EX1为fam激发光的峰值，EM1为fam发射光的峰值，EX1为cy5.5激发光的峰值，EM1为cy5.5发射光的峰值；2个不同频段的激发光包括激发光第一频段和激发光第二频段，所述激发光第一频段、激发光第二频段、发射光第一频段和发射光第二频段的频段之间互相不重合或部分重合。即光谱仪可同时检测fam激发光、fam发射光和cy5.5激发光和cy5.5发射光，其中，荧光标记物质可为fam、hex、cy5、cy5.5等，如下表所示其中， 光源为LD光源。当为LD光源时，可同时检测fam和cy5.5的荧光标记的被检测样品，而cy5、cy5.5的荧光标记的被检测样品不能被同时检测，fam、hex荧光标记的被检测样品也不能被同时检测。
其中，如fam和cy5.5的荧光标记的被检测样品，fam荧光标记的激发波长为494nm，发射波长为518nm，cy5.5荧光标记的激发波长为675nm，发射波长为695nm，即该fam荧光标记的激发波长和发射波长和cy5.5荧光标记的激发波长和发射波长无完全重合的频段。
如：激发光的第一频段范围是450-480nm，而激发光的第二频段的范围是540-560nm，即激发光的第一频段范围与激发光的第二频段范围是完全无重合的频段范围。
激发光的第一频段范围是520-550nm，而激发光的第二频段的范围是540-560nm，即激发光的第一频段范围与激发光的第二频段范围是部分无重合的频段范围。
其中，本发明提供的激发光光源可为LD光源的波长、荧光标记物质fam、hex、cy5、cy5.5的激发波长和发射波长的列表，如下表1所示。
表1为LD光源的波长、荧光标记物质fam、hex、cy5、cy5.5的激发波长和发射波长的列表；
Figure PCTCN2021071820-appb-000006
作为优选实施方式，所述光谱仪检测至少2个不同频段的激发光的频段和强度，所述光谱仪检测至少2个不同频段的激发光和由激发光照射检测样品引起的发射光的频段和强度。
所述至少2个不同频段的激发光频段至少包括激发光第一频段和激发光第二频段，2个不同频段的激发光引起的发射光的频段至少包括发射光第一频段和发射光第二频段；所述激发光第一频段、激发光第二频段、发射光第一频段和发射光第二频段的频段两两之间均无相互重合的频段范围。
至少2个不同频段的激发光的激发光频段之间无相互重合的频段范围，所至少2个不同频段的激发光引起的所述发射光的不同发射光频段之间无相互重合的频段范围；各个激发光频段以及因激发光引起的各个发射光频段之间无相互重合的频段范围。
作为优选实施方式，本发明提供的所述芯片装置包括加样层和管路层，所述加样层、管路层从上至下依次设置，所述加样层包括加样孔和试剂管，所述加样孔用于添加样品，所述试剂管用于输送buffer溶液，所述管路层包括反应仓，所述反应仓内预埋有冻干试剂，所述冻干试 剂包含荧光标记物质，其中，加样孔内的样品和buffer溶液混合后进入反应仓，从而得到被检测样品。其中buffer溶液为缓冲溶液，缓冲溶液指的是由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。
作为优选实施方式，本发明提供的激发光源模块的还包括光源旋转装置，所述光源旋转装置用于使设置有多个光发射单元PCB板旋转，即本发明的PCB板上可设置光源旋转装置，使得因激发光引起的发射光的光斑在反应仓的分布均匀，使得检测部检测更加准确。
参阅图5所示，其为本发明实施例的光源-芯片-光谱仪光路图示意图；本实施例系统包括芯片装置30、激发光模块6、温控模块4以及检测部5，所述激发光模块6发出预设波长的激发光至所述芯片装置，发生反应后，通过荧光检测部5获取荧光信息完成检测，所述温控模块4控制所述芯片装置内的温度，以使得芯片装置内的试剂达到最佳的反应状态。在本实施例中在进行扩增反应时，所述检测部5设置在所述芯片装置的反应仓上方。
参阅图6所示，本发明实施例提供的发射光检测方法所应用的装置包括：激发光源模块6、被检测样品21、检测部5，光发射单元照射所述被检测样品21，用以激发所述被检测样品21，以使所述检测部5检测所述被检测样品产生的发射光；所述被检测样品21包含荧光基团，用以在激发光的激发下产生发射光；所述检测部设置在所述发射光的光路上，用以捕捉并检测所述发射光；具体而言，在图6中示出了光路图，而激发光的光路和发射光的光路采用不同的箭头进行了表示，以示两种光的不同。
具体地，本领域技术人员可以理解的是检测部6的作用是捕捉并检测发射光，在实际应用过程中，光谱仪可为荧光光谱仪，其允许同时读取多个发射光和激发光，从而可以进行检测。其中，本发明中提供的光谱仪不包括滤光片，因此，可以同时检测多个不同频段的激发光和因激发光引起的发射光。如光谱仪可为滨松的C12889MA。
作为优选实施方式，所述激发光源模块包括多个光发射单元，所述光发射单元为发光二极管或半导体激光器。
所述激发光源模块包括PCB板、光纤合束器、至少一个发光二极管以及至少一根与每一个发光二极管对应的光纤；
发光二极管设置于PCB板一侧；在每一发光二极管的输出端设置有一光纤耦合器，每一光纤耦合器分别与一光纤耦合，通过光纤耦合器将对应波长的激发光耦合于光纤中，并通过光纤传输；
光纤合束器将各个光纤按预设方式排列合束为一体，在所述的光纤合束器的输出端还设置有光纤准直器，用以将光纤内的激发光转变成准直光；
所述至少一个发光二极管用于发射至少2个不同频段的激发光，所述至少2个不同频段的激发光的频率范围无相互重合的频率范围。
具体地，如图7所示，本发明与现有技术的激发光源模块区别在于，本发明提供的激发光源模块不包含滤光片63，而现有技术中的激发光源模块必须包含滤光片63；具体如图7所示，本发明提供的光学激励部件包括激发光源模块，激发光模块6采用体积较小的发光模块，通过采用开关对激发源模块中的激发光源进行切换，以产生不同波长的激发光。激发光模块6包括PCB板61，用以承载激发光源，如LED光源或LD光源，还可以是其他光源，LED光源设置在PCB板的一侧，在实施例中，LED光源包括若干设置的LED灯62，在每个LED灯的前端设置有一个光纤耦合器64，每一光纤耦合器64分别与一光纤65耦合，通过光纤耦合器64将对应波长的激发光耦合于光纤中，并通过光纤传输；还包括光纤合束器66，其将各个光纤按预设方式排列合束为一体，在所述光纤合束器66的输出端还设置有光纤准直器67，用以将光纤内的激发光转变成准直激发光。
作为优选实施方式，本发明提供的所述激发光源模块的还包括光源旋转装置，所述光源旋转装置用于使设置有多个光发射单元PCB板旋转，即光源旋转装置可以使PCB板旋转，从而带动光发射单元即LED光源或LD光源旋转，即本发明的PCB板上可设置光源旋转装置，使得因激发光引起的发射光的光斑在反应仓的分布均匀，使得检测部检测更加准确。
其中如图12-13所示，图12为本发明提供的激发光源模块在没有旋转的情况下，即没有调整的情况下，荧光标记物质在反应仓112上的分布情况，即分布不均匀，甚至可能不能分布在反应仓上，或荧光标记物质并没有分布在反应仓上；如图12所示，图13为本发明提供的光源旋转装置调整后的荧光标记物质在反应仓112上的分布情况，因光源旋转装置调整后，荧光标记物质在反应仓112的分布情况分布均匀，且均能分布在同一反应仓中，解决现有技术分布不均匀的技术问题，如图13所示。
激发光源模块工作过程：启动LED灯62，发出LED光源，并经过光纤耦合器将对应波长的激发光源导入光纤中，多个光纤通过光纤合束器66汇聚，并将其中传递的激发光源合为一束，并最终通过光纤准直器将传输光转变成准直激发光照射至目标，进行检测；其中LED灯为多个，可以同时发出不同波长的激发光，且不需要滤光片，本发明使用光谱仪可以识别不同波长的发射光，因此不需要滤光片。
其中，参图8所示，本发明提供的反应器为芯片装置，具体地，本发明的芯片装置包括设置在最上端的加样层3、设置在加样层3下侧的垫片2、设置在垫片2下侧的管路层101，以及设置在最下侧的密封膜104，其中，所述加样层3上侧设置有加样孔302，用以向芯片内添加样品，注入芯片内的样品经过提取、纯化、扩增发生反应。其中，本实施例的加样层与管路层通过卡条304与设置在管路层101侧部的限位架106活动连接，相应的，在限位架106的内侧设置有第一卡槽107，第一卡槽通过卡条相互配合连接，以实现加样层和管路层的相对位置切换和固定。其中，密封膜粘贴在管路层101的下侧，以实现密封。
本发明的第一卡槽107的下侧的限位架侧面上还设置有第二卡槽109，相应的，垫片2的侧壁上还设置有第二卡条(图中未示出)，通过第二卡条与第二卡槽的相互配合连接，以实现垫片2与管路层101的滑动连接，两者能够相对位置切换以及固定。本发明实施例的垫片2的下侧还设置有第一滑轨202，相应的，在管路层101的上侧面设置有第二滑槽108，第一滑轨202通过与第二滑槽108配合连接，以实现垫片与管路层101的滑动连接，两者能够相对位置切换以及固定。本实施例的第二滑槽108设置在管路层上的限位架106的内侧。所述垫片2的端部设置若干相间排列的凹口与凸起，其中，所述第一滑轨202设置在最外侧凸起的底面上。
参阅图8所示，本发明的加样孔上设置有加样孔盖303，用以进行密封。在加样层和管路层还设置卡扣结构，在加样层的一侧设置有第一卡扣301，第一卡扣301的下侧伸出端伸出所述加样层的底端，在将加样层和管路层配合安装在一起后，通过第一卡扣卡接在管路层的侧面上，以防止加样层和管路层分离。本实施例的管路层上设置有两个第一单阀102，用以分别向管路中注入样品及反应试剂；在管路层上还设置有双阀103，用以注入试剂及样品，双阀103通过管路与反应仓112连通，结合图5所示，在所述垫片2的两侧还设置有把手201，方便对芯片装置进行提取。在本发明实施例中，所述反应仓设置在管路层的边缘，并且，反应仓为半椭圆形结构，既能够使反应试剂反应，又能够在使用时，能够通过凸出的半椭圆形结构实现方便定位及安装。
参图8所示，本发明的管路层上设置有一排刺针105，通过在抽取垫片后，将刺针与加样层内的试剂连通，试剂中标记的荧光序列与对应位置的核酸刺针产生互补匹配时，通过确定荧光强度最强的探针位置，获得一组序列完全互补的探针序列。在所述刺针的外侧还设置有一挡板，其在加样层与管路层配合时，起到阻挡及定位作用。
具体而言，在本发明实施例中，在加样状态时，所述加样层内设置有若干组试剂管，所述加样层3通过其上的卡条304与第一卡槽107卡接，在初始安装状态时，加样层3自上而下与 管路层101配合，通过垫片2将刺针与试剂管内的试剂隔离，防止在运输过程中的振动造成刺针与试剂混合，避免刺破。在需要进行试验时，将垫片2沿第二滑槽108向外抽出，垫片2沿第二滑槽向外抽出后，向下按压加样层3，使得加样层上的卡条304与第二卡槽109卡接，此时，设置在管路层上的刺针与加样层的试剂混合，将试剂引入管路层内进行测定。
具体而言，本发明通过设置垫片结构，使得芯片装置能够在储存试剂，运输过程中，完好保存，在使用时，只需将垫片抽出，即能够将试剂引入管路层中。
参图9所示，其为本发明实施例的加样层的结构示意图；在本实施例的加样孔302的下方为加样仓，加样仓能够连接一装载试剂的试剂管，在加样仓的下部设置有试剂出口312，在试剂出口312与加样仓之间设置有密封结构，用以进行密封。在所述加样仓的一侧还设置有加压结构，其包括管壁305，在管壁内部设置有活塞308，活塞308向加样仓移动，推动其内的试剂向试剂出口流出；在所述活塞308的活塞杆端部设置有密封圈311，用以进行密封。
参图9所示，本实施例的活塞杆上还设置有螺帽307，通过与螺帽307螺纹连接，实现相对旋转运动，相应的，在活塞杆的一端设置有输出结构，如气缸，油缸，也可通过转动输出结构连接活塞杆，如电机、丝杠，此时，活塞杆做旋转运动，只需能够推动试剂向试剂出口流出即可。相应的，在螺帽的外侧套设有一导向套306，管壁内侧设置有相应的轴肩，用以对导向套306进行定位及固定；在导向套306的两端外侧还设置有卡环314，用以卡住相应的导向套306。在导向套306的外侧还设置有护套309，用以对活塞杆、螺帽、以及导向套进行保护。在对管路层进行试剂注射时，通过活塞向加样仓移动，增加其内的压力，以推动试剂向试剂出口流动，实现注入试剂，而在实际应用过程中，为了配合试剂的注入，提高试剂注入效率，还可以通过其他试剂管的向外吸，配合当前实际管的向内推，实现试剂的高效注入。本发明实施例设置若干组试剂管，在本实施例中，设置五组试剂管，向管路层施加试剂，试剂可以是裂解液、洗脱液或清洗液等，能够大大提高使用效率。
参图9所示，在加样层下方设置第二卡扣310，第二卡扣设置在与第一卡扣相对的一侧面上，来防止加样层滑动。
参阅图10所示，其为本发明实施例的管路层的结构示意图，本实施例的管路层设置有所述反应仓112、第一缓冲仓110、第二缓冲仓111以及对样品进行纯化的纯化仓114，其中，双阀的第一端通过第一管路118、第二管路115与纯化仓114连接；双阀的第二端与第二缓冲仓111连通，第二缓冲仓111通过管路与第一缓冲仓110连接，第一缓冲仓和第二缓冲仓之间的管路上还设置有一管路分支，该管路分支上设置有第二单阀，该管路分支另一端连接在所述 纯化仓114上，第一缓冲仓通过第三管道116、第四管道117与纯化仓114连接；在管路层上还设置有若干连接孔119，以进行连接。
具体而言，所述第一管路118包括竖向管路与横向管路，通过长距离输送洗脱后的核酸物质进入扩增仓，第二管路115包括竖向管路与横向管路，其一端与纯化仓连接，另一端与第一管路连为一体。
具体而言，所述第三管路116，其为多向弯折管，其一端与第一缓冲仓连接，另一端与第一进液口连接；所述第四管路117的一端与第一进液口连接，另一端与纯化仓连接，在第四管路117上还设置有进样口与第一单阀。
具体而言，进样口用以加入样品，第一进液口用以加入裂解液，然后打开第一单阀，使得样品和裂解液进行在混合反应，在混合过程中，可以利用与样品口连接的活塞杆和与第一进液口连接的活塞杆进行推吸操作，实现样品与裂解液的充分混合，生成第一反应物，第一反应物为液体，所述液体通过第四管路117进入所述纯化仓内，纯化仓内置磁珠，纯化仓是进行核酸提取和纯化的反应仓，然后关闭第一单阀，打开第二单阀，向所述第二进液口注入第二试剂，第二试剂为清洗液，所述第二试剂经过第二试剂口连接的管路进入所述纯化仓内，对纯化仓内的物质进行清洗，向第三进液口注入第三试剂，第三试剂为清洗液，所述第三试剂经过所述与其连接的管路进入所述纯化仓内，对纯化仓内的核酸物质进行再次清洗，向第四进液口注入第四试剂，第四试剂为洗脱液，所述第四试剂经过与其连接的管路进入所述纯化仓内，并将纯化仓内的核酸物质由其设置的磁珠上洗脱，得到核酸物质，将所述核酸物质经过第一管路115和第二管路118引入所述反应仓112内，以进行扩增反应。本发明在对管路层进行试剂注射时，通过活塞向加样仓移动，增加其内的压力，以推动试剂向试剂出口流动，实现试剂或样品的注入；本发明设置若干组活塞结构，定时向管路层施加样品或者试剂，能够大大提高使用效率。所述扩增仓设置在管路层的边缘，并且，反应仓112为半椭圆形结构，既能够使反应试剂反应，又能够在使用时，能够通过凸出的半椭圆形结构实现方便定位及安装，其中芯片装置是透明的。
尤其，本发明设置垫片及相关连接结构，一方面能够使得加样层与管路层之间能够完好的连接，避免产生震动，另一方面能够使得刺针具有较好的放置空间，垫片以及加样层滑动安装，方便拆卸。在初始安装状态时，加样层自上而下与管路层配合，通过垫片将刺针与试剂管内的试剂隔离，防止在运输过程中的振动造成刺针与试剂混合，避免刺破。在需要进行试验时，将垫片沿第二滑槽向外抽出，垫片沿第二滑槽向外抽出后，向下按压加样层，使得加样层上的卡条与第二卡槽卡接，此时，设置在管路层上的刺针与加样层的试剂混合，将试剂引入光路层内 进行测定。本发明通过设置垫片结构，使得芯片装置能够在储存试剂，运输过程中，完好保存，在使用时，只需将垫片抽出，即能够将试剂引入管路层中。
如图2所示，本发明提供的图2为单通道fam扩增前后荧光值-发射峰对比图；其中，Cycle0为未扩增的荧光光谱曲线，cycle 45为扩增了45个循环后的荧光光谱曲线；如图3所示，本发明提供的图3为fam通道荧光值与温度循环示意图；其中横坐标为时间，纵坐标为fam通道发射峰处的荧光值，由其可看出荧光曲线随温度的变化趋势，F5为荧光值随时间的变化曲线，F6为温度随时间的变化曲线，图3引出图4按照cycle取荧光值的曲线，图4为本发明提供的Fam通道按cycle取发射峰荧光值的曲线，采光的时间点在PCR扩增过程的延伸阶段结束前3s，得到曲线后可进行一种PCR仪检测方法的数据处理流程；
具体为，一种PCR检测方法，包括：
原始数据
由滨松微型光谱仪采集的cycle 0～40荧光强度
A i,i∈[0,40]
扣除基底
A 1～A 40作为信号，减去基底A 0
B i＝A i-A 0,i∈[1,40]
平滑数据
以matlab自带函数smoothdata(参数method：sgolay、winsize：9)对数据进行平滑处理，
Figure PCTCN2021071820-appb-000007
或以Savitzky-Golay平滑算法函数sg_smooth去除基底的数据B i平滑处理，其中参数为窗口大小winsize:3，阶数degree:6；
B i→C i
确定基线baseline
选择C 3～C 10(索引可选)，根据最小二乘法拟合出直线作为基线baseline
y i＝ax i+b,i∈[0,40]
归一化
数据扣除baseline
D i＝C i-y i,i∈[1,40]
一阶差分
Figure PCTCN2021071820-appb-000008
二阶差分
Figure PCTCN2021071820-appb-000009
三阶差分
D″′ i＝D″′ i+1-D″ i,i∈[4,36]
取二阶差分最大值(计算二阶差分的最大值，可以通过计算其三阶差分来得到)
具体为：选出三阶差分中符合“正负负负”或“正负负”或“正负”规律的最大点(要从二阶差分中找最大点，可以比较该点的对应的三阶差分的前中后各点)，且要求对应cycle一阶差分大于0(三种规律依次筛选，前一种成功筛选则不进行下一种筛选；反之，再进行)，该坐标+1即为二阶差分最大值对应坐标，另二阶差分最大值对应坐标±1共计3点；
多项式拟合
将上述三点按二次函数(y＝ax 2+bx+c)拟合，取
Figure PCTCN2021071820-appb-000010
切线求Ct值
过X处做D i拟合曲线的切线，其与baseline交点的横坐标即为Ct值，进而可得threshold阈值
Figure PCTCN2021071820-appb-000011
阴/阳性判别
Ct值≥38，判断为阴性；反之，则为阳性
绘图
将D i按cycle数绘制如图11中曲线。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均包含在本发明的保护范围之内。

Claims (13)

  1. 一种PCR检测仪，包括激发光源模块、芯片装置和检测部；激发光源模块、芯片装置和检测部，所述激发光源模块发出预设波长的激发光至所述芯片装置，在进行扩增反应时，所述检测部设置在所述芯片装置的反应仓一侧；
    所述芯片装置包括反应仓，反应仓内能够容纳被检测样品；其中，被检测样品为含有荧光标记的核酸片段溶液；
    所述激发光源模块用于发射激发光，激发光源模块发射的激发光能够照射置于反应仓的所述被检测样品；
    所述激发光源模块能够发射至少2个不同频段的激发光；
    其特征在于，其中，所述反应仓设置于所述激发光模块的激发光发射方向，所述检测部设置于反应仓的一侧，所述激发光照射所述反应仓后形成的发射光能够被检测部检测；其中，激发光的发射方向位于检测部的下方，所述检测部用于检测被检测样品因受激发光的照射发出的在垂直方向上的发射光；
    所述检测部包括光谱仪；所述光谱仪检测光谱的波长范围是340-850nm；所述光谱仪能够检测到所述激发光和所述发射光。
  2. 如权利要求1所述的PCR检测仪，其特征在于，所述光谱仪检测至少2个不同频段的激发光的频段和强度，所述光谱仪检测至少2个不同频段的激发光和由激发光照射检测样品引起的发射光的频段和强度。
  3. 如权利要求2所述的PCR检测仪，其特征在于，所述至少2个不同频段的激发光频段至少包括激发光第一频段和激发光第二频段，2个不同频段的激发光引起的发射光的频段至少包括发射光第一频段和发射光第二频段；所述激发光第一频段、激发光第二频段、发射光第一频段和发射光第二频段的频段两两之间均无相互重合的频段范围。
  4. 如权利要求3所述的PCR检测仪，其特征在于，所述激发光源模块包括多个光发射单元，所述光发射单元为发光二极管或半导体激光器。
  5. 如权利要求4所述的PCR检测仪，其特征在于，光发射单元为LD光源或LED光源。
  6. 如权利要求4所述的PCR检测仪，其特征在于，所述激发光源模块包括PCB板、光纤合束器、至少一个发光二极管以及至少一根与每一个发光二极管对应的光纤；
    发光二极管设置于PCB板一侧；在每一发光二极管的输出端设置有一光纤耦合器，每一光纤耦合器分别与一光纤耦合，通过光纤耦合器将对应波长的激发光耦合于光纤中，并通过光纤传输；
    光纤合束器将各个光纤按预设方式排列合束为一体，在所述的光纤合束器的输出端还设置有光纤准直器，用以将光纤内的激发光转变成准直光；
    所述至少一个发光二极管用于发射至少2个不同频段的激发光，所述至少2个不同频段的激发光的频率范围无相互重合的频率范围。
  7. 如权利要求1所述的PCR检测仪，其特征在于，所述芯片装置包括加样层和管路层，所述加样层、管路层从上至下依次设置，所述加样层包括加样孔和试剂管，所述加样孔用于添加样品，所述试剂管用于输送buffer溶液，所述管路层包括反应仓，所述反应仓内预埋有冻干试剂，所述冻干试剂包含荧光标记物质，其中，加样孔内的样品和buffer溶液混合后进入反应仓，从而得到被检测样品。
  8. 如权利要求1所述的PCR检测仪，其特征在于，所述的荧光标记的物质为fam、hex、cy5、cy5.5的至少两种，其中cy5、cy5.5荧光标记的被检测样品不能被同时检测，fam、hex荧光标记的被检测样品也不能被同时检测。
  9. 一种PCR检测方法，其特征在于，包括：
    S1：采用光谱仪采集cycle 0-40荧光强度A i，A i,i∈[0,40]；
    S2：将采集的cycle 0-40荧光强度A i作归一化处理，得到归一化处理后的数据D i，D i＝C i-y i,i∈[1,40]；y i为基线baseline，C i为平滑处理后的数据；
    S3：对步骤S2得到归一化处理后的数据D i依次作一阶差分、二阶差分和三阶差分处理；
    S4：取二阶差分最大值，选出三阶差分中符合“正负负负”或“正负负”或“正负”规律的最大点，且要求对应cycle一阶差分大于0，坐标+1即为二阶差分最大值对应坐标，另±1共计3点；
    S5：多项式拟合，将步骤S4得到的3点按二次函数y＝ax 2+bx+c拟合，取
    Figure PCTCN2021071820-appb-100001
    其中a是二次项系数，b是一次项系数，c是常数；
    S6：切线求Ct值，过X处做D i拟合曲线的切线，其与基线baseline交点的横坐标即为Ct值，进而可得threshold，
    Figure PCTCN2021071820-appb-100002
    S6：阴/阳性判别：
    若Ct值≥38，判断为阴性；反之，则为阳性。
  10. 如权利要求9所述的PCR检测方法，其特征在于，还包括步骤S7：绘图，将D i按cycle数绘制曲线。
  11. 如权利要求9所述的PCR检测方法，其特征在于，采集的cycle 0-40荧光强度A i归一化处理方法具体包括：
    S11：扣除基底：A 1-A 40作为信号，减去基底A 0；
    B i＝A i-A 0,i∈[1,40]，其中，B i为去除基底的数据；
    S12：平滑数据：对去除基底的数据B i进行平滑处理，得到平滑处理后的数据C i；
    B i→C i
    S13：确定基线：选择C 3-C 10，根据最小二乘法拟合出直线作为基线baseline y i，
    y i＝a 1χ i+b 1，i∈[1,40]，其中，a 1为一次项系数，b 1为常数项；
    S14：数据扣除基线baseline，得到D i＝C i-y i,i∈[1,40]。
  12. 如权利要求11所述的PCR检测方法，其特征在于，以Savitzky-Golay平滑算法函数sg_smooth去除基底的数据B i平滑处理，其中参数为窗口大小winsize:3，阶数degree:6。
  13. 如权利要求9所述的PCR检测方法，其特征在于，一阶差分、二阶差分和三阶差分处理具体分别为：
    S31：一阶差分
    Figure PCTCN2021071820-appb-100003
    其中，D′ i为第i个点的1阶差分值，D i+1为归一化处理后的第i+1个点，D i-1为归一化处理后的第i-1个点；
    S32：二阶差分：
    Figure PCTCN2021071820-appb-100004
    其中D″ i为第i个点二阶差分值，D′ i+2为一阶差分值中的第i+2个点的值，D′ i-2为一阶差分值中的第i-2个点的值；
    S33：三阶差分：
    D″′ i＝D″ i+1-D″ i,i∈[4,36]，其中D″′ i为第i个点的三阶差分值，D″ i+1为二阶差分中的第i+1个点的值。
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