WO2019061960A1 - 基于微流控芯片的基因检测系统及检测方法 - Google Patents

基于微流控芯片的基因检测系统及检测方法 Download PDF

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WO2019061960A1
WO2019061960A1 PCT/CN2018/073324 CN2018073324W WO2019061960A1 WO 2019061960 A1 WO2019061960 A1 WO 2019061960A1 CN 2018073324 W CN2018073324 W CN 2018073324W WO 2019061960 A1 WO2019061960 A1 WO 2019061960A1
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seq
sample
detecting
microfluidic chip
primer sequence
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PCT/CN2018/073324
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English (en)
French (fr)
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顾大勇
夏贇
何建安
徐云庆
刘春晓
史蕾
赵纯中
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深圳国际旅行卫生保健中心
深圳市检验检疫科学研究院
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Publication of WO2019061960A1 publication Critical patent/WO2019061960A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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 invention relates to the field of biotechnology, in particular to a gene detection system and a detection method based on a microfluidic chip.
  • a gene detection system based on a microfluidic chip comprising:
  • the microfluidic chip disposed in the accommodating cavity, wherein the microfluidic chip is provided with a sample adding cell, a sample dividing component and a reaction component, wherein the sample adding cell is used for adding a sample to be detected, and the sample component is Included in the arcuate channel and a plurality of decimation buffer cells, the arcuate channel being in communication with the sample application cell, the plurality of decimation buffer cells being located outside the arcuate channel and along the circumference of the arcuate channel Arranging in order, and the decimation buffer pool extends outward from the outer circumference of the arcuate passage along a radial direction of the arcuate passage, the plurality of decimation buffer pools being equal in volume, and from the The depth of the sample buffer pool is sequentially decreased from the inlet end to the outlet end of the arcuate passage, and the reaction element includes a reaction tank loaded with a bacteria detecting agent, a reaction tank loaded with a rickettsial detection agent, and a virus detecting agent.
  • An optical device comprising an excitation light source, an excitation light transmission mirror and an optical sensor, wherein the excitation light source is for emitting a laser light, and the excitation light transmission mirror is configured to focus the laser light onto the reaction cell to be detected to stimulate Detecting reactants in the reaction cell to generate an optical signal, the optical sensor for receiving the optical signal;
  • a method for detecting the content of highly pathogenic pathogenic microorganisms comprising the following steps:
  • the sample to be tested is added to a gene detection system, and the sample to be detected is placed in the sample application tank;
  • the microfluidic chip is driven to rotate at a first rate by the rotating device, so that the sample to be detected in the loading pool is sequentially entered from the arcuate channel into a plurality of the sample buffer pools;
  • the microfluidic chip is driven to rotate at a second rate by the rotating device to pass the sample to be detected in the sample buffer pool into the reaction cell through the capillary;
  • the microfluidic chip is driven to rotate at a third rate by the rotating device, so that each of the reaction cells sequentially passes through the optical device, and the laser light emitted by the excitation light source is focused and irradiated by the excitation light transmitting mirror.
  • FIG. 1 is a schematic structural view of a detection system according to an embodiment
  • Figure 2 is a schematic view of the other direction of the detection system shown in Figure 1;
  • Figure 3 is a schematic view showing a part of the structure of the detecting system shown in Figure 1;
  • Figure 4 is a schematic view showing a part of the structure of the detecting system shown in Figure 1;
  • Figure 5 is a schematic view showing a part of the structure of the detecting system shown in Figure 1;
  • Figure 6 is a schematic illustration of a portion of the structure of the detection system of Figure 1.
  • Figure 7 is a PCR amplification curve obtained by detecting the different concentrations of Bacillus anthracis using the first group of detector fluorescent PCR, and a standard curve prepared;
  • Figure 8 is a PCR amplification curve obtained by detecting the different concentrations of Brucella by the second group of detector fluorescent PCR, and a standard curve prepared;
  • Figure 9 is a PCR amplification curve obtained by detecting a different concentration of B. sinensis by fluorescent detection of a third group of detectors, and a standard curve prepared;
  • Figure 10 is a PCR amplification curve obtained by detecting a different concentration of T. mobilis using a fourth group of detector fluorescent PCR, and a standard curve prepared;
  • Figure 11 is a PCR amplification curve obtained by detecting the different concentrations of Salmonella using the fifth group of detector fluorescent PCR, and a standard curve prepared;
  • Figure 12 is a PCR amplification curve obtained by detecting the different concentrations of Salmonella typhimurium by the sixth group of detector fluorescent PCR, and a standard curve prepared;
  • Figure 13 is a PCR amplification curve obtained by detecting a different concentration of Shigella by a Group 7 detector fluorescent PCR, and a prepared standard curve;
  • qPCR amplification curve prepared by detecting changes in fluorescence intensity in each reaction cell with time when a nucleic acid sample of the rpoB gene is detected in the first example
  • Figure 15 is a flow diagram of a method of detecting the content of a highly pathogenic pathogenic microorganism in an embodiment.
  • the microfluidic chip-based gene detection system 01 of an embodiment includes a housing 10 , a microfluidic chip 20 , an optical device 30 , and a rotating device 40 .
  • the housing 10 is provided with a receiving cavity 1001, and the microfluidic chip 20 is disposed in the receiving cavity 1001.
  • the microfluidic chip 20 is substantially circular.
  • the microfluidic chip 20 is provided with a sample loading tank 210, a sample separation element 220, and a reaction element 230.
  • One of the set of loading cells 210, the decimation element 220 and the detecting element 230 form a microfluidic unit 21.
  • the microfluidic chip 20 of the present embodiment includes four microfluidic cells 21 that are evenly distributed around the center of the circle.
  • the microfluidic chip 20 can also be other shapes such as a rectangle, a polygon, and the like.
  • the number of microfluidic units 21 on the microfluidic chip 20 may also be one, two, three, five, seven, and the like.
  • the sample loading tank 210 is provided with a sample insertion hole 2001 that communicates with the outside.
  • the sorting element 220 includes an arcuate channel 221 and a plurality of decimation buffer cells 223 that communicate with the loading cell 210.
  • the plurality of sample buffer pools 223 are located outside the arcuate passages 221 and are sequentially arranged along the circumferential direction of the arcuate passages 221, and the sample buffer pools 223 are oriented from the outer circumference of the arcuate passages 221 in the radial direction of the arcuate passages 221. Extend outside.
  • the volume of the plurality of sample buffer pools 223 is equal, and the depth of the sample buffer pool 223 is sequentially decreased from the inlet end to the outlet end of the arcuate passage 221.
  • the reaction element 230 includes a plurality of reaction cells 231 loaded with a detection reagent, and the reaction cell 231 is connected to the sample buffer pool 223 through a capillary tube 240.
  • each of the sorting elements 220 includes 16 equal-sized sample buffer pools 223, and the number of reaction cells 231 is matched with the sample buffer pool 223.
  • the entire microfluidic chip 20 is provided with 64 equal-sized sample buffer pools 223 and 64 reaction cells 231 for high-throughput detection.
  • the sample buffer pool 223 is a rectangular sample buffer pool, and the bottom of the rectangular sample buffer pool is chamfered.
  • the sample to be detected after centrifugation is allowed to enter the reaction cell 231 without residue, and the sample actually participating in the reaction is more accurate.
  • the aspect ratio of the sample buffer pool 223 is 1:1 to 4:1, and the aspect ratio of the inlet end closest to the curved channel 210 is larger, and the aspect ratio of the exit end closest to the curved channel 210 is smaller.
  • the depth refers to the distance from the inlet end to the bottom of the sample buffer tank 223, and the width refers to the width of the opening of the sample buffer tank 223.
  • the aspect ratio of the inlet end closest to the curved passage 210 is 4:1, and the aspect ratio of the outlet end closest to the curved passage 210 is 1:1.
  • the sample to be tested can smoothly fill each of the sample buffer pools 223, and the sample to be tested in the sample buffer pool 223 is equal in volume.
  • the sorting element 220 further includes a waste liquid tank 225 disposed at an outlet end of the curved passage 210, and the waste liquid tank 225 extends radially outward of the curved passage 210.
  • the sample to be tested is sequentially filled with a plurality of sample buffer pools 223 from the inlet end to the outlet end of the curved channel 210, and the excess sample to be tested flows into the waste liquid pool 225, and the sample loading process is convenient and quick.
  • the reaction element 230 includes a plurality of reaction cells 231 loaded with a detection agent, such as a reaction cell 231 loaded with a bacteria detecting agent, a reaction cell 231 loaded with a rickettsial detection agent, a reaction cell 231 loaded with a virus detecting agent, and a fungus loaded with fungi.
  • a detection agent such as a reaction cell 231 loaded with a bacteria detecting agent, a reaction cell 231 loaded with a rickettsial detection agent, a reaction cell 231 loaded with a virus detecting agent, and a fungus loaded with fungi.
  • the reaction tank 231 of the detection agent and the reaction tank 231 loaded with the biotoxin detecting agent.
  • the distance from the bottom of each reaction cell 231 to the edge of the microfluidic chip 20 is equal, for example, 1 mm.
  • the optical path propagation distance is equal when detecting, and the optical signal propagation coefficient is as small as possible.
  • the reaction element 230 includes a reaction cell 231 that is loaded with a detector, each of which includes an upstream primer, a downstream primer, and a probe.
  • a detector each of which includes an upstream primer, a downstream primer, and a probe.
  • the first group of detecting agents for detecting Bacillus anthracis, the sequence of the upstream primer is shown in SEQ ID No. 1, the downstream primer sequence is shown in SEQ ID No. 2, and the probe sequence is as shown in SEQ ID No. 3. Show. The above primers and probes were designed for the rpoB gene.
  • Group 2 detector for detection of Brucella, the upstream primer sequence is shown in SEQ ID No. 4, the downstream primer sequence is shown in SEQ ID No. 5, and the probe sequence is shown in SEQ ID No. 6.
  • the above primers and probes were designed for the IS711 gene.
  • Group 3 detection agent for detecting B. sinensis, the upstream primer sequence is shown in SEQ ID No. 7, the downstream primer sequence is shown in SEQ ID No. 8, and the probe sequence is as shown in SEQ ID No. 9. Show.
  • the above primers and probes were designed for the fliP gene.
  • Group 4 detection agent for detecting T. mobilis, the upstream primer sequence is shown in SEQ ID No. 10, the downstream primer sequence is shown in SEQ ID No. 11, and the probe sequence is shown in SEQ ID No. .
  • the above primers and probes were designed for the tul4 gene.
  • Group 5 detector for detection of Salmonella, the upstream primer sequence is shown in SEQ ID No.
  • the downstream primer sequence is shown in SEQ ID No. 14, and the probe sequence is shown in SEQ ID No. 15.
  • the above primers and probes were designed for the invA gene.
  • Group 6 detector for detecting Salmonella typhi, the upstream primer sequence is shown in SEQ ID No. 16, the downstream primer sequence is shown in SEQ ID No. 17, and the probe sequence is shown in SEQ ID No. 18.
  • the above primers and probes were designed for the staG gene.
  • Group 7 detector for detecting Shigella, the upstream primer sequence is shown in SEQ ID No. 19, the downstream primer sequence is shown in SEQ ID No. 20, and the probe sequence is shown in SEQ ID No. 21.
  • the above primers and probes were designed for the ipaH gene.
  • Group 8 detector for detection of Chlamydia psittaci, the upstream primer sequence is shown in SEQ ID No. 22, the downstream primer sequence is shown in SEQ ID No. 23, and the probe sequence is shown in SEQ ID No. 24.
  • the above primers and probes were designed for the ompA gene.
  • Group 9 detecting agent for detecting rickettsial, the upstream primer sequence is shown in SEQ ID No. 25, the downstream primer sequence is shown in SEQ ID No. 26, and the probe sequence is SEQ ID No. 27. Shown. The above primers and probes were designed for the gltA gene.
  • Group 10 detector for detecting Ebola virus, the upstream primer sequence is shown in SEQ ID No. 28, the downstream primer sequence is shown in SEQ ID No.
  • the above primers and probes are designed for Ebola virus nucleoproteins.
  • Group 11 detection agent for detecting Hantavirus, the upstream primer sequence is shown in SEQ ID No. 31, the downstream primer sequence is shown in SEQ ID No. 32, and the probe sequence is shown in SEQ ID No. 33.
  • the above primers and probes were designed against Hantavirus nucleoprotein.
  • Group 12 detector for detecting avian influenza virus, the upstream primer sequence is shown in SEQ ID No. 34, the downstream primer sequence is shown in SEQ ID No. 35, and the probe sequence is shown in SEQ ID No. 36.
  • the above primers and probes are directed against avian influenza virus matrix proteins.
  • Group 13 detector for detecting variola virus, the upstream primer sequence is shown in SEQ ID No. 37, the downstream primer sequence is shown in SEQ ID No. 38, and the probe sequence is shown in SEQ ID No. 39. The above primers and probes were designed for the A38R gene.
  • Group 14 detection agent for detecting Clostridium botulinum, the upstream primer sequence is shown in SEQ ID No. 40, the downstream primer sequence is shown in SEQ ID No. 41, and the probe sequence is as shown in SEQ ID No. 42. Show. The above primers and probes were designed for the botA gene.
  • Group 15 detector for detecting Staphylococcus aureus, the upstream primer sequence is shown in SEQ ID No.
  • the downstream primer sequence is shown in SEQ ID No. 44
  • the probe sequence is shown in SEQ ID No. 45.
  • the above primers and probes were designed for the fmhB gene.
  • Group 16 detection agent for detecting acacia toxin
  • the upstream primer sequence is shown in SEQ ID No. 46
  • the downstream primer sequence is shown in SEQ ID No. 47
  • the probe sequence is shown in SEQ ID No. 48.
  • the above primers and probes are designed for lectins.
  • the reaction element 230 further includes a reaction cell 231 that respectively loads the 17th group of detection agents.
  • the 17th group of detecting agents are used for bacterial positive control
  • the upstream primer sequence is shown in SEQ ID No. 49
  • the downstream primer sequence is shown in SEQ ID No. 50
  • the probe sequence is shown in SEQ ID No. 51.
  • the above primers and probes were designed for the 16S rDNA gene. Through the nature control, more accurate reaction detection results.
  • a FAM fluorescent group is disposed on the 5' end of the probe, and a TAMRA fluorescent group is disposed on the 3' end.
  • the annealing temperature of each primer is different, which causes the annealing temperature to be difficult to coordinate and the detection result is inaccurate.
  • specific target genes of pathogenic microorganisms were searched, and the above-mentioned detectors designed for specific genes or proteins were designed and screened.
  • the specific annealing temperature of 17 sets of detector primers was designed to be around 60 °C, avoiding simultaneous detection. Due to the different annealing temperatures, the detection results are inaccurate.
  • one reaction cell 231 is loaded with a set of detection agents.
  • the concentration of the upstream primer is 300 nmol/L to 500 nmol/L
  • the concentration of the downstream primer is 300 nmol/L to 500 nmol/L
  • the concentration of the probe is 200 nmol/L to 400 nmol/L.
  • the microfluidic chip 20 includes a bottom plate and a top plate, and a corresponding sample loading tank 210, a sample buffer pool 223, and a tank of the reaction cell 231 are opened on the bottom plate, and the groove depth is 2.0 mm.
  • the upstream primers, the downstream primers and the probes of each group are mixed and formulated into a preset liquid, and then respectively pointed into the reaction tank 231, and the two or more reaction cells 231 may contain the same detecting agent in one
  • the results of two or more parallel experiments were obtained during the test to improve the accuracy of the test.
  • the top plate is packaged, dried at room temperature or lyophilized to prepare a microfluidic chip 20.
  • the top plate is a pressure sensitive film having high transparency
  • the pressure sensitive film having high transparency at the time of detection faces the side of the optical device 30.
  • the edge of the microfluidic chip 20 is polished so that the coefficient of variation of the optical signal propagation during detection is as small as possible.
  • the capillary tube 240 includes a liquid guiding tube 241 and a blocking tube 243, and the capillary tube 240 is subjected to a hydrophobic treatment.
  • the liquid guiding tube 241 is used to connect the sample buffer pool 223 and the reaction cell 231.
  • the blocking tube 243 is crossed with the liquid guiding tube 251, and a part of the liquid guiding tube 241 extends radially outward of the liquid guiding tube 241 to form a blocking tube 243.
  • the catheter 241 and the stopper tube 243 are combined to form a shape of a cross.
  • the microfluidic chip 20 is further provided with a siphon channel 250 for communicating the sample loading tank 210 and the sample separation element 220.
  • a siphon channel 250 for communicating the sample loading tank 210 and the sample separation element 220.
  • One end of the siphon channel 250 is connected to the sample loading tank 210, and the other end is connected to the inlet end of the curved channel 210.
  • the siphon channel 250 is provided with a plurality of curved passages 251 to prevent backflow of liquid.
  • the siphon channel 250 is subjected to a hydrophilic treatment, and the siphon channel 250 draws the liquid in the loading tank 210 into the curved channel 210. Under the action of the centrifugal motion, the liquid in the curved channel 210 is sequentially filled with a plurality of sample buffer pools 223, and the excess sample to be detected flows into the waste liquid pool 225.
  • the microfluidic chip 20 is further provided with an exhaust pipe 260 for conducting the flow of the sample loading tank 210 and the sample separation element 220, and one end of the exhaust pipe 260 is connected to the sample loading tank 210. The other end of the exhaust pipe 260 is connected to the outlet end of the arcuate passage 210.
  • the exhaust pipe 260 By providing the exhaust pipe 260, the air pressure in the sample loading tank 210 and the curved passage 210 is balanced, so that the liquid in the curved passage 210 is sequentially filled with the plurality of sample buffer pools 223.
  • a part of the exhaust pipe 260 extends radially outward of the exhaust pipe 260 to form an exhaust cavity 261, and the exhaust cavity 261 is provided with a vent hole 2003 communicating with the outside.
  • the extruded gas enters the exhaust chamber 261 through the exhaust pipe 260 and is discharged by the exhaust holes 2003.
  • the volume at the exhaust chamber 261 is large to prevent liquid from being spilled.
  • the microfluidic chip 20 is provided with a heat conduction channel 270 that extends through the microfluidic chip 20. When heated, the air flow on both sides of the microfluidic chip 20 is conducted through the heat conduction channel 270, so that the microfluidic chip 20 is heated uniformly.
  • the microfluidic chip 20 is a circular microfluidic chip, and the sample loading tank 210, the sample separation component 220 and the reaction component 300 are sequentially distributed outward along the radial direction of the microfluidic chip 20, and the curved channel 210 and the microflow
  • the control chip 20 is arranged concentrically.
  • the optical device 30 includes an excitation light source 310 , an excitation light transmission mirror 320 , an emission light lens 330 , a filter 340 , and an optical sensor 350 .
  • the excitation light source 310 is used to emit laser light
  • the excitation light transmission mirror 320 is used to focus the laser light onto the reaction cell 231 to be detected to excite the reactants in the reaction cell 231 to be detected to generate an optical signal.
  • the excitation light source 310 is, for example, a light emitting diode or a laser light source or the like.
  • the reactants in the reaction cell 231 generate an optical signal under the illumination of the excitation light
  • the emission optical lens 330 is used to concentrate the optical signal generated by the reactants in the reaction cell 231.
  • the filter 340 is used to filter the converged optical signals for transmission to the optical sensor 350, and the signal intensity of each of the reaction cells 231 is received and recorded by the optical sensor 350.
  • the optical sensor 350 is, for example, a photomultiplier tube or a photodiode or the like.
  • the exiting light lens 330 and the filter 340 may also be omitted.
  • the laser light emitted by the excitation source 310 is incident on the reaction cell 231 to be detected, and the laser is perpendicular to the plane in which the microfluidic chip 20 is located.
  • the excitation light source 310 is facing the microfluidic chip 20, and the emitted laser light is irradiated on the reaction cell 231.
  • the optical sensor 350 is mounted on the housing 10, and the optical sensor 350 is parallel to the plane in which the microfluidic chip 20 is located.
  • the optical signals emitted by the respective reaction cells 231 are received during the rotation of the microfluidic chip 20.
  • the excitation light source 310, the excitation light transmission mirror 320, and the reaction cell 231 to be detected are in a straight line.
  • the reaction cell 231, the exiting light lens 330, the filter 340, and the optical sensor 350 to be detected are in a straight line. Further, the exiting optical lens 330, the filter 340, and the optical sensor 350 are disposed adjacent to the edge of the microfluidic chip 20.
  • the line connecting the excitation light source 310, the excitation light transmitting mirror 320 and the reaction cell 231 to be detected is connected with the reaction cell 231 to be detected, the light exiting lens 330, the filter 340 and the optical sensor 350 in a reaction cell to be detected.
  • the intersection of 231 and the two straight lines are perpendicular to each other, which reduces the difference caused by the instrument during the detection and improves the sensitivity of the detection.
  • the rotating device 40 is configured to drive the microfluidic chip 20 to rotate, so that each of the reaction cells 231 sequentially passes through the optical device 30 to automate the detection process.
  • the turning device 40 is, for example, a rotating electrical machine or the like.
  • the detection system 01 further includes a heater 50 and a chiller 60, both of which are located within the accommodating chamber 1001.
  • the heater 50 includes a heat source 51 and a heat sink 51 for providing thermal energy.
  • the heat sink 51 is disposed around the heat source 51.
  • the heat sink 53 is, for example, a fan, and the heat generated by the heat source 51 is accelerated by the heat sink 53 to be diffused into the accommodating chamber 1001, so that the temperature of the microfluidic chip 20 is uniform.
  • the cooler 60 is used to cool the microfluidic chip 20. During the reaction, depending on the process, it is necessary to rapidly switch the temperature so that the sample to be detected and the detecting agent can react smoothly in the reaction cell 231. Specifically, the cooler 60 is disposed directly under the microfluidic chip 20.
  • the heater 50 and the cooler 60 provided above realize the synchronous rapid temperature control of the high-throughput microfluidic chip by the non-contact balance temperature control technology.
  • the temperature control technology has the following advantages: a) reduced temperature control cost, no longer depends on the traditional contact thermoelectric semiconductor module, and is more suitable for small micro flow The overall temperature control of the control chip; b) rapid temperature rise and temperature, and the uniformity of the heat transfer temperature of the rotary air is better; c) the compatibility of the expansion is strong, and the microfluidic chip of different shapes and structures can perform the temperature well. Control and optimization tests eliminate the size limitations of traditional contact temperature control modules and have no edge effects.
  • the housing 10 is provided with an air passage switch 1003.
  • the housing 10 is hermetically sealed, and the gas passage switch 1003 is opened or closed to accelerate the convection of the accommodating chamber 1001 with the outside air.
  • the microfluidic chip 20 is detachably disposed on the rotating device 40.
  • the detection system 01 also includes a locking mechanism 70 that locks the microfluidic chip 20, through which the locking mechanism 70 prevents the microfluidic chip 20 from deviating from the running track during centrifugation.
  • the above detection system 01 can simultaneously detect a plurality of pathogenic microorganisms at one time, the sample processing steps are simple, the detection efficiency is high, and the sample loading amount is easy to control, the volume of the sample to be detected entering each reaction cell 231 is equal, and the detection result is more accurate.
  • the detection process is automated to meet the application requirements of high efficiency and large sample size of pathogenic microorganisms for rapid detection and inspection of port health and quarantine.
  • a method for detecting the content of a highly pathogenic pathogenic microorganism includes the following steps S110 to S150.
  • the method can be used to detect or diagnose diseases, and can also be used for non-disease diagnosis and treatment.
  • the structure of the detection system can be referred to FIG. 1 to FIG. 6 and the above description, and details are not described herein.
  • the sample to be tested may be serum, powder or the like.
  • the sample to be tested is formulated into a solution form, and is added from the sample introduction hole 2001 to the sample introduction tank 210.
  • S120 The microfluidic chip is driven to rotate at a first rate by the rotating device, so that the sample to be detected in the loading pool is sequentially entered into the plurality of the sampling buffer pools from the curved channel.
  • the first rate is from 800 rpm to 1000 rpm.
  • the sample to be detected enters the curved channel 210 by centrifugation, and a plurality of sample buffer pools 223 are sequentially filled from the inlet end to the outlet end of the curved channel 210, and the excess sample to be detected flows into the waste liquid pool 225.
  • the microfluidic chip is driven to rotate at a second rate by a rotating device to pass the sample to be detected in the sample buffer pool into the reaction cell through the capillary.
  • the second rate is 2500 rpm to 3000 rpm.
  • the sample to be detected in the sample buffer pool 223 passes through the capillary 240 into the reaction cell 231, and reacts with the detector previously stored in the reaction cell 231.
  • the third rate is 200 rpm to 600 rpm.
  • the microfluidic chip 20 is rotated at a lower rate, and the reaction cell 231 is sequentially passed through the optical device 30, and the corresponding reaction light signal is detected by the optical device 30.
  • the optical sensor records the fluorescence signal intensity values during the reaction of all the reaction cells 231, draws a corresponding amplification curve, and analyzes the reaction results to obtain the content of each highly pathogenic pathogenic microorganism in the sample to be tested.
  • the sample to be detected is added to the loading tank of the microfluidic chip, and the microfluidic chip is mounted on the rotating device, and the rotating device drives the microfluidic chip to perform the first rotation, and the sample to be detected
  • the arc channel is filled, and a plurality of sample buffer pools are sequentially filled from the inlet end to the outlet end of the arc channel.
  • the plurality of sample buffer pools are equal in volume and the depth from the inlet end of the arc channel to the outlet end sample buffer pool is sequentially decreased, so that the sample to be tested can smoothly fill each sample buffer pool to ensure the waiting in the sample buffer pool.
  • the sample volume is tested to be equal.
  • the microfluidic chip is driven to rotate a second time by the rotating device, and the sample to be detected in the sampling buffer pool enters the reaction cell from the capillary and reacts with the detecting agent loaded in the reaction cell.
  • each reaction cell passes through the optical device in turn, and the laser light emitted from the excitation light source is focused and irradiated onto the reaction cell to be detected through the excitation light transmission mirror, and the reactants in the reaction cell to be detected are excited.
  • the optical signal is generated, and the optical sensor receives the optical signal, thereby calculating parameters such as the content of each highly pathogenic pathogenic microorganism in the sample to be detected.
  • the above detection method can directly detect untreated complex samples, such as serum, powder, etc., and has the ability to rapidly detect multiple pathogens in a single reaction, shorten the health quarantine response time of the port, and improve the detection efficiency.
  • the above detection method is highly automated and avoids potential bio-safety threats.
  • the centrifugal microfluidic chip technology can quickly achieve high-throughput distribution of samples of the same biological sample or clinical sample, reducing reagent consumption and time consumption, and having significant cost. benefit.
  • Test Example 1 Microfluidic Chip High-throughput Gene Detection System 01
  • the reaction element 230 includes 64 reaction cells 231, numbered from No. 1 to No. 64, respectively.
  • No. 1 to No. 3 were loaded with the first group of detection agents at three concentrations of high, medium and low
  • No. 4 to No. 51 were loaded with the second to the sixth group of detection agents at three concentrations of high, medium and low.
  • Group 52 was loaded with the 17th group of detectors (positive control).
  • Nos. 53-64 are blank controls.
  • the concentration of the upstream primer of the medium concentration detecting agent was 400 nmol/L
  • the concentration of the downstream primer was 400 nmol/L
  • the concentration of the probe was 300 nmol/L
  • the concentration of the upstream primer of the low concentration detecting agent was 300 nmol/L
  • the concentration of the downstream primer was 300 nmol/L
  • the concentration of the probe was 200 nmol/L.
  • the concentration of the upstream primer in the 17th detection agent was 400 nmol/L
  • the concentration of the downstream primer was 400 nmol/L
  • the concentration of the probe was 300 nmol/L.
  • a preliminary experiment was carried out in which a different concentration of Bacillus anthracis was detected by the first group of detector fluorescent PCR, and the obtained PCR amplification curve and the prepared standard curve are shown in FIG. 7 .
  • Different concentrations of Brucella were detected by the second group of detector fluorescent PCR, and the obtained PCR amplification curves and the prepared standard curve are shown in FIG.
  • the third group of detector fluorescent PCR was used to detect different concentrations of B. sinensis, and the obtained PCR amplification curve and the prepared standard curve are shown in FIG. 9 .
  • the fourth group of detector fluorescent PCR to detect different concentrations of T. mobilis, the obtained PCR amplification curve and the prepared standard curve are shown in FIG.
  • the different concentrations of Salmonella were detected by the fifth group of detector fluorescent PCR, and the obtained PCR amplification curve and the prepared standard curve are shown in FIG.
  • the different concentrations of Salmonella typhimurium were detected by the sixth group of detector fluorescent PCR, and the obtained PCR amplification curve and the prepared standard curve are shown in FIG.
  • the different concentrations of Shigella were detected by the 7th detection agent fluorescent PCR, and the obtained PCR amplification curve and the prepared standard curve are shown in FIG.
  • the concentration of the upstream primer in each group of detection agents was 400 nmol/L
  • the concentration of the downstream primer was 400 nmol/L
  • the concentration of the probe was 300 nmol/L. It can be seen from Fig. 7 to Fig.
  • the sample to be detected (nucleic acid sample of the rpoB gene) is then added from the sample well 2001 to the sample addition tank 210.
  • the loaded microfluidic chip 20 is placed on the rotating device 40 and locked by a locking mechanism 70.
  • the detection system 01 is activated, and the rotating device 40 drives the microfluidic chip 20 to centrifuge at 800 rpm for 1 min, and the sample is centrifuged to enter the curved channel 210, and a plurality of sample buffer pools 223 are sequentially filled from the inlet end to the outlet end of the curved channel 210. .
  • the plurality of sample buffer pools 223 are equal in volume and the depth from the inlet end of the curved channel 210 to the outlet end sample buffer pool 223 is sequentially decreased, so that the samples to be tested can smoothly fill each of the sample buffer pools 223, and the sample separation is ensured.
  • the volume of the sample to be detected in the buffer pool 223 is equal.
  • the rotating device 40 drives the microfluidic chip 20 to centrifuge at 2500 rpm for 2 min, so that the sample to be detected in each of the sample buffer pools 223 enters the reaction cell 231 from the capillary 240, and reacts with the detecting agent previously stored in the reaction cell 231. .
  • the centrifugation rate was then reduced to 400 rpm.
  • the heater 50 is started to be heated, and the heat source 51 starts to heat up to heat the air in the accommodation chamber 1001, while the radiator 53 starts to operate and causes the inner circulation airflow to agitate the hot air so that the microfluidic chip 20 is uniformly heated.
  • the heat source 51 will reduce the power to maintain the denaturation temperature required for the microfluidic chip 20 to perform PCR (polymerase chain reaction).
  • the gas passage switch 1003 is opened and the heat source 51 is stopped.
  • the cooler 60 starts to work, and the external cold air is introduced into the system to replace the original hot air to complete the cooling.
  • the gas passage switch 1003 is closed, and the cooler 60 stops working, and the microfluidic chip 20 is maintained for PCR.
  • the optical device 30 completes the fluorescence detection of the reaction cell 231 during the PCR extension time of the microfluidic chip 20.
  • the excitation light source 310 starts to work, and the reaction cell of the microfluidic chip 20 at this time 231 will sequentially pass the optical path of the excitation light at a rotation speed of 400 rpm, and the excitation light is focused on the reaction cell 231 by the excitation light transmitting mirror 320 and excites the detection agent (such as a primer and a probe) in the reaction cell 231 for qPCR reaction.
  • the fluorescent signal produced by the hydrolysis probe.
  • the fluorescent signal passes through the exiting optical lens 330 and the filter 340 in sequence, and is finally received by the optical sensor 350.
  • the optical sensor 350 accepts and records the current cyclic signal intensity.
  • the system After 40 cycles, the system records the 40 rounds of circulating fluorescent signal intensity values of all the reaction cells 231, and draws a corresponding qPCR amplification curve. The result is shown in FIG. There were qPCR amplification curves in the four reaction cells No. 1 to No. 3 and No. 52, while other numbered reaction cells had no qPCR amplification curve.
  • the detection system 01 can obtain the light signals of the respective reaction cells 231, and simultaneously detect a plurality of pathogenic microorganisms, and the sample processing steps are simple and the detection efficiency is high.

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Abstract

提供了一种基于微流控芯片的基因检测系统及检测方法。该基因检测系统包括壳体、微流控芯片、光学装置及转动装置。微流控芯片设置在容纳腔内，微流控芯片上设有加样池、分样元件以及反应元件，反应元件包括多个装载检测剂的反应池。

Description

基于微流控芯片的基因检测系统及检测方法 技术领域
本发明涉及生物技术领域，特别是涉及一种基于微流控芯片的基因检测系统及检测方法。
背景技术
高致病性病原微生物大多数具有感染力强、传播快、潜伏期短和发病急等特点，所引起的疾病病原学复杂，给人类的健康、社会的稳定以及畜牧业安全等带来极大的威胁。当前，一些高致病性病原微生物已经跨越了种属之间的障碍，不定期在人类中爆发成为越来越常见的现象。由于口岸卫生检疫对象的复杂性、流动性，以及潜在高致病性病原微生物的未知性，多变性等众多因素，快速检测高致病性病原微生物非常有必要。
然而，传统的检测产品自动化程度不高，检测效率低，检测结果不准确。远远不能满足口岸卫生检疫高效率、大样本量病原微生物快速检测排查的应用要求。
发明内容
基于此，有必要提供一种检测效率高、检测结果准确的基于微流控芯片的基因检测系统及检测方法。
一种基于微流控芯片的基因检测系统，包括：
壳体，内设有容纳腔；
微流控芯片，设置在所述容纳腔内，所述微流控芯片上设有加样池、分样元件以及反应元件，所述加样池用于加入待检测样品，所述分样元件包括弧形通道和多个分样缓冲池，所述弧形通道与所述加样池连通，所述多个分样缓冲池位于所述弧形通道的外侧且沿所述弧形通道的周向依次排布，且所述分样缓冲池沿所述弧形通道的径向自所述弧形通道的外周缘向外延伸，所述多个分样缓冲池的体积相等，且从所述弧形通道的进口端至出口端方向所述分样缓冲池的深度依次减小，所述反应元件包括装载细菌检测剂的反应池、装载立克次体检测剂的反应池、装载病毒检测剂的反应池、装载真菌检测剂的反应池和装载生物毒素检测剂的反应池，所述反应池与所述分样缓冲池通过毛细管连通；
光学装置，包括激发光源、激发光透射镜和光学传感器，所述激发光源用于发射激光，所述激发光透射镜用于将所述激光聚焦照射到待检测的所述反应池上，以激发待检测的所述反应池内的反应物产生光信号，所述光学传感器用于接收所述光信号；及
转动装置，用于带动所述微流控芯片转动，以使各个所述反应池依次经过所述光学装置。
一种检测高致病性病原微生物含量的方法，包括如下步骤：
将待检测样品加入基因检测系统中，所述待检测样品置于所述加样池中；
通过所述转动装置带动所述微流控芯片以第一速率进行离心转动，以将所述加样池内的所述待检测样品从所述弧形通道依次进入多个所述分样缓冲池内；
通过所述转动装置带动所述微流控芯片以第二速率进行离心转动，以将所述分样缓冲池内的所述待检测样品通过所述毛细管进入所述反应池内；
通过所述转动装置带动所述微流控芯片以第三速率进行离心转动，使各个所述反应池依次经过所述光学装置，所述激发光源发射的激光通过所述激发光透射镜聚焦照射到待检测的所述反应池上，以激发待检测的所述反应池内的反应物产生光信号，所述光学传感器接收所述光信号；以及
根据所述光信号计算获得所述待检测样品中各高致病性病原微生物含量。
本发明的一个或多个实施例的细节在下面的附图和描述中提出。本发明的其它特征、目的和优点将从说明书、附图以及权利要求书变得明显。
附图说明
图1为一实施方式的检测系统的结构示意图；
图2为图1所示的检测系统另一个方向的示意图；
图3为图1所示检测系统的部分结构的示意图；
图4为图1所示检测系统的部分结构的示意图；
图5为图1所示检测系统的部分结构的示意图；
图6为图1所示检测系统的部分结构的示意图。
图7为用第1组检测剂荧光PCR检测不同的浓度的炭疽芽孢杆菌，获得的PCR扩增曲线以及制得的标准曲线图；
图8为用第2组检测剂荧光PCR检测不同的浓度的布鲁氏杆菌，获得的PCR扩增曲线以及制得的标准曲线图；
图9为用第3组检测剂荧光PCR检测不同的浓度的鼻疽伯克氏菌，获得的PCR扩增曲线以及制得的标准曲线图；
图10为用第4组检测剂荧光PCR检测不同的浓度的土拉弗氏菌制，获得的PCR扩增曲线以及制得的标准曲线图；
图11为用第5组检测剂荧光PCR检测不同的浓度的沙门氏菌，获得的PCR扩增曲线以及制得的标准曲线图；
图12为用第6组检测剂荧光PCR检测不同的浓度的伤寒沙门氏菌，获得的PCR扩增曲线以及制得的标准曲线图；
图13为用第7组检测剂荧光PCR检测不同的浓度的志贺氏菌，获得的PCR扩增曲线以及制得的标准曲线图；
图14为检测例一中检测rpoB基因的核酸样本时各反应池中荧光强度随时间变化制得的qPCR扩增曲线；
图15为一实施方式的检测高致病性病原微生物含量的方法的流程图。
具体实施方式
下面结合具体实施例及附图对本发明的具体实施方式做详细的说明。
请参阅图1和图2，一实施方式的基于微流控芯片的基因检测系统01，包括壳体10、微流控芯片20、光学装置30以及转动装置40。其中壳体10内设有容纳腔1001，微流控芯片20设置在容纳腔1001内。
一实施方式的微流控芯片20的结构请参阅图3和图4，该微流控芯片20大致呈圆形。微流控芯片20上设有加样池210、分样元件220和反应元件230。其中一组加样池210、分样元件220和检测元件230形成一个微流控单元21。本实施方式的微流控芯片20包括四个绕圆心均匀分布的微流控单元21。当然，在其他实施方式中，微流控芯片20还可以是其他形状，例如矩形、多边形等等。微流控芯片20上的微流控单元21的数量还可以为一个、两个、三个、五个、七个等等。
具体地，加样池210上设有与外界连通的加样孔2001。分样元件220包括弧形通道221和多个分样缓冲池223，弧形通道221与加样池210连通。多个分样缓冲池223位于弧形通道221的外侧且沿弧形通道221的周向依次排布，且分样缓冲池223沿弧形通道221的径向自弧形通道221的外周缘向外延伸。多个分样缓冲池223的体积相等，且从弧形通道221的进口端至出口端方向分样缓冲池223的深度依次减小。反应元件230包括多个装载检测剂的反应池231，反应池231与分样缓冲池223通过毛细管240连通。具体地，每个分样元件220包括16个体积相等的分样缓冲池223，反应池231的数量与分样缓冲池223匹配。整个微流控芯片20上设有64个体积相等的分样缓冲池223和64个反应池231，实现高通量的检测。
具体地，分样缓冲池223为矩形分样缓冲池，矩形分样缓冲池的池底设有倒角。使得经过离心后待检测样品无残留的进入反应池231中，实际参与反应的样品更加准确。具体地，分样缓冲池223的深宽比为1:1～4:1，最靠近弧形通道210的进口端的深宽比越大，最靠近弧形通道210的出口端的深宽比越小。深是指分样缓冲池223的进口端至底部的距离，宽是指分样缓冲池223开口的宽度。在本实施方式中，最靠近弧形通道210的进口端的深宽比为4:1，最靠近弧形通道210的出口端的深宽比为1:1。待检测样品能够顺利填满每个分样缓冲池223，保证分样缓冲池内223的待检测样品体积相等。
具体地，分样元件220还包括废液池225，废液池225设置在弧形通道210的出口端，废液池225 沿弧形通道210的径向向外延伸。经过离心后，待检测样品从弧形通道210的进口端至出口端依次填充多个分样缓冲池223，多余的待检测样品流入废液池225中，加样过程方便快捷。
具体地，反应元件230包括多个装载检测剂的反应池231，例如装载细菌检测剂的反应池231、装载立克次体检测剂的反应池231、装载病毒检测剂的反应池231、装载真菌检测剂的反应池231和装载生物毒素检测剂的反应池231。每个反应池231底部到微流控芯片20边缘距离相等，例如为1mm。使得检测时光路传播距离相等，光信号传播变异系数尽可能小。
在一个实施方式中，反应元件230包括分别装载如下检测剂的反应池231，每一组检测剂中均包括上游引物、下游引物和探针。其中，第1组检测剂：用于检测炭疽芽孢杆菌，上游引物的序列如SEQ ID No.1所示，下游引物序列如SEQ ID No.2所示，探针序列如SEQ ID No.3所示。上述引物和探针针对rpoB基因设计。第2组检测剂：用于检测布鲁氏杆菌，上游引物序列如SEQ ID No.4所示，下游引物序列如SEQ ID No.5所示，探针序列如SEQ ID No.6所示。上述引物和探针针对IS711基因设计。第3组检测剂：用于检测鼻疽伯克氏菌，上游引物序列如SEQ ID No.7所示，下游引物序列如SEQ ID No.8所示，探针序列如SEQ ID No.9所示。上述引物和探针针对fliP基因设计。第4组检测剂：用于检测土拉弗氏菌，上游引物序列如SEQ ID No.10所示，下游引物序列如SEQ ID No.11所示，探针序列如SEQ ID No.12所示。上述引物和探针针对tul4基因设计。第5组检测剂：用于检测沙门氏菌，上游引物序列如SEQ ID No.13所示，下游引物序列如SEQ ID No.14所示，探针序列如SEQ ID No.15所示。上述引物和探针针对invA基因设计。第6组检测剂：用于检测伤寒沙门氏菌，上游引物序列如SEQ ID No.16所示，下游引物序列如SEQ ID No.17所示，探针序列如SEQ ID No.18所示。上述引物和探针针对staG基因设计。第7组检测剂：用于检测志贺氏菌，上游引物序列如SEQ ID No.19所示，下游引物序列如SEQ ID No.20所示，探针序列如SEQ ID No.21所示。上述引物和探针针对ipaH基因设计。第8组检测剂：用于检测鹦鹉热衣原体，上游引物序列如SEQ ID No.22所示，下游引物序列如SEQ ID No.23所示，探针序列如SEQ ID No.24所示。上述引物和探针针对ompA基因设计。第9组检测剂：用于检测普氏立克次氏体，上游引物序列如SEQ ID No.25所示，下游引物序列如SEQ ID No.26所示，探针序列如SEQ ID No.27所示。上述引物和探针针对gltA基因设计。第10组检测剂：用于检测埃博拉病毒，上游引物序列如SEQ ID No.28所示，下游引物序列如SEQ ID No.29所示，探针序列如SEQ ID No.30所示。上述引物和探针针对埃博拉病毒核蛋白设计。第11组检测剂：用于检测汉坦病毒，上游引物序列如SEQ ID No.31所示，下游引物序列如SEQ ID No.32所示，探针序列如SEQ ID No.33所示。上述引物和探针针对汉坦病毒核蛋白设计。第12组检测剂：用于检测禽流感病毒，上游引物序列如SEQ ID No.34所示，下游引物序列如SEQ ID No.35所示，探针序列如SEQ ID No.36所示。上述引物和探针针对禽流感病毒基质蛋白。第13组检测剂：用于检测天花病毒，上游引物序列如SEQ ID No.37所示，下游引物序列如SEQ ID No.38所示，探针序列如SEQ ID No.39所示。上述引物和探针针对A38R基因设计。第14组检测剂：用于检测肉毒梭状芽孢杆菌，上游引物序列如SEQ ID No.40所示，下游引物序列如SEQ ID No.41所示，探针序列如SEQ ID No.42所示。上述引物和探针针对botA基因设计。第15组检测剂：用于检测金黄色葡萄球菌，上游引物序列如SEQ ID No.43所示，下游引物序列如SEQ ID No.44所示，探针序列如SEQ ID No.45所示。上述引物和探针针对fmhB基因设计。第16组检测剂：用于检测相思子毒素，上游引物序列如SEQ ID No.46所示，下游引物序列如SEQ ID No.47所示，探针序列如SEQ ID No.48所示。上述引物和探针针对凝集素设计。具体地，反应元件230还包括分别装载第17组检测剂的反应池231。其中第17组检测剂：用于细菌阳性质控，上游引物序列如SEQ ID No.49所示，下游引物序列如SEQ ID No.50所示，探针序列如SEQ ID No.51所示。上述引物和探针针对16S rDNA基因设计。通过阳性质控，更加准确的反应检测结果。
具体地，探针的5'端上设有FAM荧光基团，3'端上设有TAMRA荧光基团。在检测的过程中，如果同时针对多种病原微生物进行检测，常出现因各个引物退火温度不同，导致同时检测时退火温度难以协调，检测结果不准确的问题。本研究查找具有特异性的病原微生物靶标基因，设计筛选出上述分别针对特定的基因或蛋白进行设计的检测剂，特异性设计的17组检测剂引物退火温度均在60℃左 右，避免同时检测时因退火温度不同导致检测结果不准确的问题，经过一次样本处理，能够同时检测7种细菌、2种立克次体、4种病毒、1种真菌和2种生物毒素，以及1个阳性质控，检测准确性好，灵敏度高。从未具有统一扩增条件，实现大规模阵列qPCR，简化基于传统扩增管单一样本检测的实现难度，提高qPCR检测通量与检测效率，符合口岸卫生检疫的实际需求。
具体地，一个反应池231装载一组检测剂。一组检测剂中，上游引物的浓度为300nmol/L～500nmol/L，下游引物的浓度为300nmol/L～500nmol/L，探针的浓度为200nmol/L～400nmol/L。
具体地，微流控芯片20包括底板和顶板，在底板上开设相应的加样池210、分样缓冲池223和反应池231的槽，槽深均为2.0mm。将每一组的上游引物、下游引物、探针混合配制成预置液，然后分别点到反应池231中，两个或两个以上的反应池231中可以含有相同的检测剂，以在一个检测中获得两个或两个以上的平行实验的结果，提高检测的准确性。点样完成后用顶板封装，常温干燥或冻干，制得微流控芯片20。具体地，顶板为透明度高的压敏膜，检测的时候透明度高的压敏膜朝向光学装置30一侧。具体地，微流控芯片20的边缘进行了抛光处理，使得检测时光信号传播变异系数尽可能小。
具体地，请参阅图5，本实施方式中，毛细管240包括导液管241和阻止管243，毛细管240进行了疏水处理。导液管241用于连通分样缓冲池223和反应池231。阻止管243与导液管251交叉，部分导液管241延导液管241的径向向外凸起形成阻止管243。具体地，导液管241和阻止管243组合形成十字架的形状。当待检测样品经过第二次离心进入反应池231中后，后续加热反应时，进入反应池231的溶液不会倒流入分样缓冲池223中造成液体泄漏以及交叉污染。
在一个实施方式中，微流控芯片20上还设有虹吸通道250，虹吸通道250用于连通加样池210和分样元件220。该虹吸通道250的一端连接加样池210，另一端连接弧形通道210的进口端，虹吸通道250上设有多个弯道251，避免液体倒流。具体地，虹吸通道250进行亲水处理，虹吸通道250将加样池210中的液体吸入到弧形通道210中。在离心运动的作用下，弧形通道210内的液体依次填充多个分样缓冲池223，多余的待检测样品流入废液池225中。
在一个实施方式中，微流控芯片20上还设有排气管260，排气管260用于将加样池210和分样元件220气流导通，排气管260一端连接加样池210，排气管260的另一端连接弧形通道210的出口端。通过设置排气管260，使得加样池210和弧形通道210内的气压平衡，便于弧形通道210内的液体依次填充多个分样缓冲池223。具体地，部分排气管260延排气管260的径向向外凸起形成排气腔261，排气腔261上设有与外界连通的排气孔2003。待检测样品填充多个分样缓冲池223后，挤出的气体通过排气管260进入排气腔261中，由排气孔2003排出。排气腔261处的体积较大，以防液体被溅出。
在一个实施方式中，微流控芯片20上设有导热通道270，导热通道270贯穿微流控芯片20。在加热时，通过导热通道270导通微流控芯片20两面的气流，使得微流控芯片20受热均匀。
具体的，微流控芯片20为圆形微流控芯片，加样池210、分样元件220和反应元件300沿微流控芯片20的径向依次向外分布，弧形通道210与微流控芯片20同心设置。
具体地，请参阅图6，光学装置30包括激发光源310、激发光透射镜320、射出光透镜330、滤片340以及光学传感器350。激发光源310用于发射激光，激发光透射镜320用于将激光聚焦照射到待检测的反应池231上，以激发待检测的反应池231内的反应物产生光信号。激发光源310例如为发光二极管或激光光源等。反应池231中的反应物在激发光的照射下产生光信号，射出光透镜330用于汇聚反应池231内的反应物产生的光信号。滤片340用于将汇聚后的光信号过滤以传输至光学传感器350中，通过光学传感器350接受并记录每个反应池231的信号强度。光学传感器350例如为光电倍增管或光电二极管等。当然，在其他实施方式中，射出光透镜330和滤片340也可以省略。
在一个实施方式中，激发光源310发射的激光照射在待检测的反应池231上，且激光与微流控芯片20所在的平面垂直。激发光源310正对微流控芯片20，发射的激光照射在反应池231上。具体地，光学传感器350安装在壳体10上，且光学传感器350与微流控芯片20所在的平面平行。在微流控芯 片20转动的过程中接受各个反应池231发出的光信号。具体地，检测时，激发光源310、激发光透射镜320和待检测的反应池231三者在一条直线上。待检测的反应池231、射出光透镜330、滤片340以及光学传感器350在一条直线上。进一步地，射出光透镜330、滤片340以及光学传感器350靠近微流控芯片20的边缘设置。激发光源310、激发光透射镜320和待检测的反应池231连成的直线与待检测的反应池231、射出光透镜330、滤片340以及光学传感器350连成的直线在待检测的反应池231处相交且两条直线相互垂直，降低检测时因仪器导致的差异性，提高检测的灵敏度。
具体地，转动装置40用于带动微流控芯片20转动，以使各个反应池231依次经过光学装置30，实现检测过程自动化。转动装置40例如为旋转电机等。
请再次参阅图1，在一个实施方式中，检测系统01还包括加热器50和冷却器60，加热器50和冷却器60均位于容纳腔1001内。加热器50包括热源51和散热器53，热源51用于提供热能。散热器51环绕热源51设置。散热器53例如为风扇，通过散热器53加速热源51产生的热量扩散到容纳腔1001内，使得微流控芯片20升温均匀。冷却器60用于对微流控芯片20降温。在反应过程中，根据进程不同，需要快速切换温度，以使待检测样品与检测剂在反应池231内能够顺利反应。具体地，冷却器60设置在微流控芯片20的正下方。
上述设置的加热器50和冷却器60，利用非接触平衡温控技术实现高通量微流控芯片的同步快速温度控制。该温控技术相较于传统基于热电半导体(帕尔贴)的加热方式具有以下几个优势：a)减少了温度控制成本，不再依赖于传统接触式热电半导体模块，更适合于小型微流控芯片的整体温度控制；b)升降温迅速，且旋转式空气传热温度均一性更好；c)扩展兼容性强，针对不同形状及不同结构的微流控芯片都能很好的进行温度控制及优化测试，摆脱了传统接触式温控模块的尺寸局限且没有边缘效应。
在一个实施方式中，壳体10上设有空气通道开关1003。壳体10气密封好，打开或关闭气体通道开关1003，加速容纳腔1001与外界的气体对流。
具体的，微流控芯片20可拆卸的设置在转动装置40。检测系统01还包括锁定微流控芯片20的锁定机构70，通过的锁定机构70防止离心过程中微流控芯片20偏离运行轨道。
上述检测系统01，一次上样能够同时检测多种病原微生物，样品处理步骤简单，检测效率高，同时加样量容易控制，进入每个反应池231中的待检测样品体积相等，检测结果更加准确，检测过程自动化，满足口岸卫生检疫高效率、大样本量病原微生物快速检测排查的应用要求。
请参阅图15，一实施方式的的检测高致病性病原微生物含量的方法，包括如下步骤S110～S150。该方法可用于检测或诊断疾病，也能够用于非疾病诊断和治疗。
S110、将待检测样品加入检测系统中，其中待检测样品置于加样池中。
具体地，检测系统的结构可参见图1～图6以及上文描述，在此不作赘述。具体地，待检测样品可以是血清，粉末等。将待检测样品配制成溶液形式，从加样孔2001加入到加样池210中。
S120、通过转动装置带动微流控芯片以第一速率进行离心转动，以将加样池内的待检测样品从弧形通道依次进入多个所述分样缓冲池内。
具体地，第一速率为800rpm～1000rpm。通过离心使得待检测样品进入弧形通道210，从弧形通道210的进口端至出口端依次填充多个分样缓冲池223，多余的待检测样品流入废液池225中。
S130、通过转动装置带动微流控芯片以第二速率进行离心转动，以将分样缓冲池内的待检测样品通过毛细管进入反应池内。
具体地，第二速率为2500rpm～3000rpm。在较大的离心速率作用下，分样缓冲池内223的待检测样品经过从毛细管240进入反应池231中，与预先储存在反应池231内的检测剂发生反应。
S140、通过转动装置带动微流控芯片以第三速率进行离心转动，使各个反应池依次经过光学装置，激发光源发射的激光通过激发光透射镜聚焦照射到待检测的反应池上，以激发待检测的反应池内的反应物产生光信号，光学传感器接收光信号。
具体地，第三速率为200rpm～600rpm。微流控芯片20以较低的速率转动，反应池231依次经过光学装置30，通过光学装置30检测获得相应的反应光信号。
S150、根据光信号计算获得待检测样品中各高致病性病原微生物含量。
例如，光学传感器记录所有反应池231反应过程中的荧光信号强度值，绘制对应的扩增曲线，并对反应结果进行分析，获得待检测样品中各高致病性病原微生物含量。
在一个实施方式中，将待检测样品加入到微流控芯片的加样池中，并将微流控芯片安装在转动装置上，转动装置带动微流控芯片进行第一次转动，待检测样品在离心作用下进入弧形通道，从弧形通道的进口端至出口端依次填充多个分样缓冲池。多个分样缓冲池体积相等且从弧形通道的进口端至出口端分样缓冲池的深度依次减小，便于待检测样品顺利填满每个分样缓冲池，保证分样缓冲池内的待检测样品体积相等。然后通过转动装置带动微流控芯片进行第二次转动，分样缓冲池内的待检测样品从毛细管进入反应池中，与装载在反应池内的检测剂发生反应。之后微流控芯片在过转动装置的转动过程中，各个反应池依次经过光学装置，激发光源发射的激光通过激发光透射镜聚焦照射到待检测的反应池上，激发待检测的反应池内的反应物产生光信号，光学传感器接收光信号，从而计算获得待检测样品中各高致病性病原微生物含量等参数。
上述检测方法能够直接检测未经处理的复杂样本，例如血清，粉末等，且具有单次反应同步快速检测多种病原体的能力，缩短口岸卫生检疫响应时间，提高检测效率。上述检测方法自动化程度高，避免了潜在的生物安全威胁，通过离心微流控芯片技术可快速实现同一生物样本或临床样本的样品高通量分配，减少了试剂消耗与时间消耗，具有显著的成本效益。
以下为具体检测例
检测例一：微流控芯片高通量基因检测系统01如图1所示，反应元件230包括64个反应池231，标号分别为1号～64号。其中1号～3号分别装载高、中、低三个浓度的第1组检测剂，4号～51号分别装载高、中、低三个浓度的第2组～第16组检测剂。52号装载第17组检测剂(阳性对照)。第53号～64号为空白对照。其中1号～51号中高浓度检测剂上游引物的浓度为500nmol/L、下游引物的浓度500nmol/L、探针的浓度为400nmol/L。中浓度检测剂上游引物的浓度为400nmol/L、下游引物的浓度400nmol/L、探针的浓度为300nmol/L。低浓度检测剂上游引物的浓度为300nmol/L、下游引物的浓度300nmol/L、探针的浓度为200nmol/L。第17组检测剂中上游引物的浓度为400nmol/L、下游引物的浓度400nmol/L、探针的浓度为300nmol/L。先进行预实验，其中，用第1组检测剂荧光PCR检测不同的浓度的炭疽芽孢杆菌，获得的PCR扩增曲线以及制得的标准曲线如图7所示。用第2组检测剂荧光PCR检测不同的浓度的布鲁氏杆菌，获得的PCR扩增曲线以及制得的标准曲线如图8所示。用第3组检测剂荧光PCR检测不同的浓度的鼻疽伯克氏菌，获得的PCR扩增曲线以及制得的标准曲线如图9所示。用第4组检测剂荧光PCR检测不同的浓度的土拉弗氏菌制，获得的PCR扩增曲线以及制得的标准曲线如图10所示。用第5组检测剂荧光PCR检测不同的浓度的沙门氏菌，获得的PCR扩增曲线以及制得的标准曲线如图11所示。用第6组检测剂荧光PCR检测不同的浓度的伤寒沙门氏菌，获得的PCR扩增曲线以及制得的标准曲线如图12所示。用第7组检测剂荧光PCR检测不同的浓度的志贺氏菌，获得的PCR扩增曲线以及制得的标准曲线如图13所示。各组检测剂中上游引物的浓度为400nmol/L，下游引物的浓度为400nmol/L，探针的浓度为300nmol/L。从图7～图13可以看出，各组检测剂制得的标准曲线线性好，说明设计的引物特异性好、灵敏度高，且各组检测剂引物退火温度均在60℃左右，避免同时检测时因退火温度不同导致检测结果不准确的问题，能够用在一块微流控芯片20上进行检测。
然后将待检测样品(rpoB基因的核酸样本)从加样孔2001加入到加样池210中。将加样后的微流控芯片20放置在转动装置40上，并用锁定机构70锁定。启动检测系统01，转动装置40带动微流控芯片20以800rpm离心1min，离心使得待检测样品进入弧形通道210，从弧形通道210的进口端至出口端依次填充多个分样缓冲池223。多个分样缓冲池223体积相等且从弧形通道210的进口端至出口端分样缓冲池223的深度依次减小，便于待检测样品顺利填满每个分样缓冲池223，保证分样缓冲池223内的待检测样品体积相等。然后转动装置40带动微流控芯片20以2500rpm离心2min，使得每个分样缓冲池223内的待检测样品从毛细管240进入反应池231中，与预先储存在反应池231内的检测剂发生反应。然后将离心速率降至400rpm。启动加热器50进行加热，热源51开始升温对容 纳腔1001内的空气进行加热，同时散热器53开始工作并引起内循环气流将热空气搅动使得微流控芯片20得到均匀加热。达到目标温度95℃后，热源51将降低功率以维持微流控芯片20进行PCR(聚合酶链式反应)所需的变性温度。此后，气体通道开关1003打开，热源51停止工作。冷却器60开始工作，将外部冷空气引入系统中替代原有热空气完成降温，达到目标温度60℃后，气体通道开关1003关闭，同时冷却器60停止工作，维持微流控芯片20进行PCR所需的延伸时间。这样即完成一轮PCR循环过程。光学装置30在微流控芯片20进行PCR延伸时间内完成对反应池231的荧光检测，当容纳腔1001内温度达到60℃时，激发光源310开始工作，此时微流控芯片20的反应池231在400rpm的转速下将依次通过激发光的光路，激发光在激发光透射镜320的作用下聚焦在反应池231中并激发出反应池231内检测剂(如引物、探针)进行qPCR反应水解探针所产生的荧光信号。荧光信号依次通过射出光透镜330和滤片340，最终被光学传感器350接收。光学传感器350接受并记录当前循环信号强度，40轮循环后，系统将记录完所有反应池231的40轮循环荧光信号强度值，绘制对应的qPCR扩增曲线，结果如图14所示。第1号～3号以及第52号四个反应池中有qPCR扩增曲线，而其他编号的反应池无qPCR扩增曲线。该检测系统01能够获得各个反应池231的光信号，同时检测多种病原微生物，样品处理步骤简单，检测效率高。
以上所述实施例仅表达了本发明的一种或几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (18)

  1. 一种基于微流控芯片的基因检测系统，包括：
    壳体，内设有容纳腔；
    微流控芯片，设置在所述容纳腔内，所述微流控芯片上设有加样池、分样元件以及反应元件，所述加样池用于加入待检测样品，所述分样元件包括弧形通道和多个分样缓冲池，所述弧形通道与所述加样池连通，所述多个分样缓冲池位于所述弧形通道的外侧且沿所述弧形通道的周向依次排布，且所述分样缓冲池沿所述弧形通道的径向自所述弧形通道的外周缘向外延伸，所述多个分样缓冲池的体积相等，且从所述弧形通道的进口端至出口端方向所述分样缓冲池的深度依次减小，所述反应元件包括装载细菌检测剂的反应池、装载立克次体检测剂的反应池、装载病毒检测剂的反应池、装载真菌检测剂的反应池和装载生物毒素检测剂的反应池，所述反应池与所述分样缓冲池通过毛细管连通；
    光学装置，包括激发光源、激发光透射镜和光学传感器，所述激发光源用于发射激光，所述激发光透射镜用于将所述激光聚焦照射到待检测的所述反应池上，以激发待检测的所述反应池内的反应物产生光信号，所述光学传感器用于接收所述光信号；及
    转动装置，用于带动所述微流控芯片转动，以使各个所述反应池依次经过所述光学装置。
  2. 根据权利要求1所述的基因检测系统，其特征在于，所述反应元件包括分别装载如下检测剂的反应池，每一组所述检测剂中均包括上游引物、下游引物和探针；
    第1组检测剂：用于检测炭疽芽孢杆菌，上游引物的序列如SEQ ID No.1所示，下游引物序列如SEQ ID No.2所示，探针序列如SEQ ID No.3所示；
    第2组检测剂：用于检测布鲁氏杆菌，上游引物序列如SEQ ID No.4所示，下游引物序列如SEQ ID No.5所示，探针序列如SEQ ID No.6所示；
    第3组检测剂：用于检测鼻疽伯克氏菌，上游引物序列如SEQ ID No.7所示，下游引物序列如SEQ ID No.8所示，探针序列如SEQ ID No.9所示；
    第4组检测剂：用于检测土拉弗氏菌，上游引物序列如SEQ ID No.10所示，下游引物序列如SEQ ID No.11所示，探针序列如SEQ ID No.12所示；
    第5组检测剂：用于检测沙门氏菌，上游引物序列如SEQ ID No.13所示，下游引物序列如SEQ ID No.14所示，探针序列如SEQ ID No.15所示；
    第6组检测剂：用于检测伤寒沙门氏菌，上游引物序列如SEQ ID No.16所示，下游引物序列如SEQ ID No.17所示，探针序列如SEQ ID No.18所示；
    第7组检测剂：用于检测志贺氏菌，上游引物序列如SEQ ID No.19所示，下游引物序列如SEQ ID No.20所示，探针序列如SEQ ID No.21所示；
    第8组检测剂：用于检测鹦鹉热衣原体，上游引物序列如SEQ ID No.22所示，下游引物序列如SEQ ID No.23所示，探针序列如SEQ ID No.24所示；
    第9组检测剂：用于检测普氏立克次氏体，上游引物序列如SEQ ID No.25所示，下游引物序列如SEQ ID No.26所示，探针序列如SEQ ID No.27所示；
    第10组检测剂：用于检测埃博拉病毒，上游引物序列如SEQ ID No.28所示，下游引物序列如SEQ ID No.29所示，探针序列如SEQ ID No.30所示；
    第11组检测剂：用于检测汉坦病毒，上游引物序列如SEQ ID No.31所示，下游引物序列如SEQ ID No.32所示，探针序列如SEQ ID No.33所示；
    第12组检测剂：用于检测禽流感病毒，上游引物序列如SEQ ID No.34所示，下游引物序列如SEQ ID No.35所示，探针序列如SEQ ID No.36所示；
    第13组检测剂：用于检测天花病毒，上游引物序列如SEQ ID No.37所示，下游引物序列如SEQ ID No.38所示，探针序列如SEQ ID No.39所示；
    第14组检测剂：用于检测肉毒梭状芽孢杆菌，上游引物序列如SEQ ID No.40所示，下游引物序列如SEQ ID No.41所示，探针序列如SEQ ID No.42所示；
    第15组检测剂：用于检测金黄色葡萄球菌，上游引物序列如SEQ ID No.43所示，下游引物序列 如SEQ ID No.44所示，探针序列如SEQ ID No.45所示；
    第16组检测剂：用于检测相思子毒素，上游引物序列如SEQ ID No.46所示，下游引物序列如SEQ ID No.47所示，探针序列如SEQ ID No.48所示。
  3. 根据权利要求2所述的基因检测系统，其特征在于，所述检测剂中，所述上游引物的浓度为300nmol/L～500nmol/L，所述下游引物的浓度为300nmol/L～500nmol/L，所述探针的浓度为200nmol/L～400nmol/L。
  4. 根据权利要求1所述的基因检测系统，其特征在于，所述光学装置还包括射出光透镜和滤片，其中，所述射出光透镜用于汇聚所述反应池内的反应物产生的所述光信号，所述滤片用于将汇聚后的所述光信号过滤以传输至所述光学传感器。
  5. 根据权利要求1所述的基因检测系统，其特征在于，所述激发光源发射的所述激光照射在待检测的所述反应池上，且所述激光与所述微流控芯片所在的平面垂直，所述光学传感器安装在所述壳体上，且所述光学传感器与所述微流控芯片所在的平面平行。
  6. 根据权利要求1所述的基因检测系统，其特征在于，所述微流控芯片上还设有：
    废液池，所述废液池设置在所述弧形通道的出口端，所述废液池沿所述弧形通道的径向向外延伸。
  7. 根据权利要求1所述的基因检测系统，其特征在于，所述微流控芯片上还设有：
    虹吸通道，用于连通所述加样池和所述分样元件，所述虹吸通道的一端连接所述加样池，所述虹吸通道的另一端连接所述弧形通道的进口端，所述虹吸通道上设有多个弯道。
  8. 根据权利要求1所述的基因检测系统，其特征在于，所述微流控芯片上还设有：
    排气管，用于将所述加样池和所述分样元件气流导通，所述排气管一端连接所述加样池，所述排气管的另一端连接所述弧形通道的出口端，部分所述排气管延所述排气管的径向向外凸起形成排气腔，所述排气腔上设有与外界连通的排气孔。
  9. 根据权利要求1所述的基因检测系统，其特征在于，所述分样缓冲池为矩形分样缓冲池，所述矩形分样缓冲池的池底设有倒角，所述分样缓冲池的深宽比为1:1～4:1。
  10. 根据权利要求1所述的基因检测系统，其特征在于，所述检测系统还包括：
    加热器，位于所述容纳腔内，所述加热器包括热源和散热器，所述热源用于提供热能，所述散热器环绕所述热源设置；及
    冷却器，位于所述容纳腔内，所述冷却器用于对所述微流控芯片降温。
  11. 一种检测高致病性病原微生物含量的方法，包括如下步骤：
    将待检测样品加入基因检测系统中，所述基因检测系统包括壳体、微流控芯片、光学装置及转动装置，所述壳体内设有容纳腔，所述微流控芯片设置在所述容纳腔内，所述微流控芯片上设有加样池、分样元件以及反应元件，所述加样池用于加入待检测样品，所述分样元件包括弧形通道和多个分样缓冲池，所述弧形通道与所述加样池连通，所述多个分样缓冲池位于所述弧形通道的外侧且沿所述弧形通道的周向依次排布，且所述分样缓冲池沿所述弧形通道的径向自所述弧形通道的外周缘向外延伸，所述多个分样缓冲池的体积相等，且从所述弧形通道的进口端至出口端方向所述分样缓冲池的深度依次减小，所述反应元件包括装载细菌检测剂的反应池、装载立克次体检测剂的反应池、装载病毒检测剂的反应池、装载真菌检测剂的反应池和装载生物毒素检测剂的反应池，所述反应池与所述分样缓冲池通过毛细管连通，所述光学装置包括激发光源、激发光透射镜和光学传感器，所述激发光源用于发射激光，所述激发光透射镜用于将所述激光聚焦照射到待检测的所述反应池上，以激发待检测的所述反应池内的反应物产生光信号，所述光学传感器用于接收所述光信号，所述转动装置用于带动所述微流控芯片转动，以使各个所述反应池依次经过所述光学装置，所述待检测样品置于所述加样池中；
    通过所述转动装置带动所述微流控芯片以第一速率进行离心转动，以将所述加样池内的所述待检测样品从所述弧形通道依次进入多个所述分样缓冲池内；
    通过所述转动装置带动所述微流控芯片以第二速率进行离心转动，以将所述分样缓冲池内的所述待检测样品通过所述毛细管进入所述反应池内；
    通过所述转动装置带动所述微流控芯片以第三速率进行离心转动，使各个所述反应池依次经过所 述光学装置，所述激发光源发射的激光通过所述激发光透射镜聚焦照射到待检测的所述反应池上，以激发待检测的所述反应池内的反应物产生光信号，所述光学传感器接收所述光信号；以及
    根据所述光信号计算获得所述待检测样品中各高致病性病原微生物含量。
  12. 根据权利要求11所述的方法，其特征在于，所述第一速率为800rpm～1000rpm，所述第二速率为2500rpm～3000rpm，所述第三速率为200rpm～600rpm。
  13. 根据权利要求11所述的方法，其特征在于，所述光学装置还包括射出光透镜和滤片，其中，所述射出光透镜用于汇聚所述反应池内的反应物产生的所述光信号，所述滤片用于将汇聚后的所述光信号过滤以传输至所述光学传感器。
  14. 根据权利要求11所述的方法，其特征在于，所述激发光源发射的所述激光照射在待检测的所述反应池上，且所述激光与所述微流控芯片所在的平面垂直，所述光学传感器安装在所述壳体上，且所述光学传感器与所述微流控芯片所在的平面平行。
  15. 根据权利要求11所述的方法，其特征在于，所述微流控芯片上还设有：
    废液池，所述废液池设置在所述弧形通道的出口端，所述废液池沿所述弧形通道的径向向外延伸。
  16. 根据权利要求11所述的方法，其特征在于，所述微流控芯片上还设有：
    虹吸通道，用于连通所述加样池和所述分样元件，所述虹吸通道的一端连接所述加样池，所述虹吸通道的另一端连接所述弧形通道的进口端，所述虹吸通道上设有多个弯道；及
    排气管，用于将所述加样池和所述分样元件气流导通，所述排气管一端连接所述加样池，所述排气管的另一端连接所述弧形通道的出口端，部分所述排气管延所述排气管的径向向外凸起形成排气腔，所述排气腔上设有与外界连通的排气孔。
  17. 根据权利要求11所述的方法，其特征在于，所述分样缓冲池为矩形分样缓冲池，所述矩形分样缓冲池的池底设有倒角，所述分样缓冲池的深宽比为1:1～4:1。
  18. 根据权利要求11所述的方法，其特征在于，所述检测系统还包括：
    加热器，位于所述容纳腔内，所述加热器包括热源和散热器，所述热源用于提供热能，所述散热器环绕所述热源设置；及
    冷却器，位于所述容纳腔内，所述冷却器用于对所述微流控芯片降温。
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