AU2020100366A4

AU2020100366A4 - Gene detection system and method based on microfluidic chip

Info

Publication number: AU2020100366A4
Application number: AU2020100366A
Authority: AU
Inventors: Dayong GU; Jianan He; Chunxiao LIU; Lei Shi; Yun Xia; Yunqing XU; Chunzhong ZHAO
Original assignee: Shenzhen Int Travel Healthcare Center Shenzhen Customs Port Outpatient Department; Shenzhen Second Peoples Hospital; Shenzhen Academy of Inspection and Quarantine
Current assignee: Shenzhen International Travel Healthcare Center Shenzhen Customs Port Outpatient Department; Shenzhen Second Peoples Hospital; Shenzhen Academy of Inspection and Quarantine
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-09-03
Anticipated expiration: 2028-03-11

Abstract

The present disclosure relates to a gene detection system and a detection method based on a microfluidic chip. The detection system includes a housing, a microfluidic chip, an optical device, and a rotating device. The microfluidic chip is disposed in a receiving cavity. The microfluidic chip is provided with a sample loading cell, a sample dispensing element, and a reaction element. The reaction element includes a plurality of reaction cells in which detection reagents are loaded. The aforementioned detection system can detect various pathogenic microorganisms at the same time with a single sample loading, has simple sample processing steps and high detection efficiency. In addition, the amount of the sample loading is easy to control, the volume of the sample to be detected entering each reaction cell is equal, and the detection results are more accurate and the detection process is automated, which meets the application requirements of high efficiency of port health quarantine and quick detection and investigation of large-sample-amount pathogenic microorganisms. 3/8 20 21 21 21 21 FIG 3 21 230 240 220 250 251 223 221 231 210 231 2001 260 231 2003 261 270 225 231

Description

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GENE DETECTION SYSTEM AND METHOD BASED ON MICROFLUIDIC CHIP TECHNICALFIELD

[0001] The present disclosure relates to the technical field of biological detections, in particular to a gene detection system and a detection method based on a microfluidic chip.

BACKGROUND

[0002] Most of highly pathogenic microorganisms have the characteristics of strong infection capacity, quick transmission, short incubation period, acute disease onset, and the like. The etiology of the caused diseases is complex, and great threats are brought to the health of human beings, the stability of the society, the safety of animal husbandry, and the like. Currently, some highly pathogenic microorganisms have spanned barriers between species, with unscheduled outbreaks in humans becoming an increasingly common phenomenon. Due to the complexity and the mobility of port health quarantine objects, and a plurality of factors such as the unknown and the variability of potential highly pathogenic microorganisms, the rapid detection of the highly pathogenic microorganisms is very necessary.

[0003] The conventional detection methods mainly include direct smear microscopy, separation culture, and the like. However, the microscopic examination method relying on the in vitro culture of the pathogenic microorganisms is long in time consumption, complex in operation, and unsatisfactory in efficiency and throughput. Detection means deep to the molecular level and the gene level are continuously appeared and widely applied. Among them, a series of detection methods developed based on Polymerase Chain Reaction (PCR) and antigen-antibody reaction are typical. However, the conventional immunoassay technology has the problems that the detection of the window period of pathogenic microorganism infection is difficult, even if the infected person is infected with virus, the result of virus antibody detection is negative and the diagnosis is missed because the copy number of the virus is small and the abundance of the virus antibody is low. The real-time fluorescent quantitative PCR technology is a mainstream method for detecting pathogenic microorganism nucleic acid molecules at present, the method has strong specificity, but only one pathogenic target can be detected in one experiment, the detection throughput is low, and meanwhile, the method cannot well cope with the detection of a large number of pathogenic targets. Both methods often take a long time to detect various pathogenic microorganisms, which delays the optimal time for diagnosis and treatment. In conclusion, the conventional detection product has low automation degree, low detection efficiency, and inaccurate detection result. The application requirements of high efficiency and large sample quantity of pathogenic microorganisms for port health quarantine on rapid detection and investigation cannot be met.

SUMMARY

[0004] Accordingly, it is necessary to provide a gene detection system and a detection method based on a microfluidic chip, which have high detection efficiency and accurate detection results.

[0005] A gene detection system based on a microfluidic chip includes:

[0006] a housing provided with a receiving cavity therein;

[0007] a microfluidic chip disposed in the receiving cavity, the microfluidic chip being provided with a sample loading cell, a sample dispensing element, and a reaction element, the sample loading cell being configured to load a sample to be detected, the sample dispensing element comprising an arc-shaped channel and a plurality of sample dispensing buffer cells, the arc-shaped channel being in communication with the sample loading cell, the plurality of sample dispensing buffer cells being located outside the arc-shaped channel and being sequentially arranged along a circumferential direction of the arc-shaped channel, the sample dispensing buffer cell extending outward from a peripheral edge of the arc-shaped channel along a radial direction of the arc-shaped channel, each of the sample dispensing buffer cells having the same volume, depths of the sample dispensing buffer cells sequentially decreasing from an inlet end of the arc-shaped channel to an outlet end thereof, the reaction element comprising a reaction cell loaded with a bacteria detection agent, a reaction cell loaded with a rickettsia detection agent, a reaction cell loaded with a virus detection agent, a reaction cell loaded with a fungus detection agent, and a reaction cell loaded with a biotoxin detection agent, the reaction cells being in communication with the sample dispensing buffer cell through a capillary tube;

[0008] an optical device comprising an excitation light source, an excitation light transmission mirror, and an optical sensor, the excitation light source being configured to emit laser, the excitation light transmission mirror being configured to focus and irradiate the laser onto the reaction cells to be detected so as to excite a reactant in the reaction cells to be detected to generate an optical signal, and the optical sensor being configured to receive the optical signal; and

[0009] a rotating device configured to drive the microfluidic chip to rotate so that each of the reaction cells sequentially goes through the optical device.

[0010] A method of detecting content of pathogenic microorganisms includes:

[0011] loading a sample to be detected in the sample loading cell of the aforementioned detection system;

[0012] driving, by the rotating device, the microfluidic chip to centrifugally rotate at a first rate, such that the sample to be detected in the sample loading cell sequentially enters the plurality of sample dispensing buffer cells through the arc-shaped channel;

[0013] driving, by the rotating device, the microfluidic chip to centrifugally rotate at a second rate, such that the sample to be detected in sample dispensing buffer cells enters the reaction cells through the capillary tube;

[0014] driving, by the rotating device, the microfluidic chip to centrifugally rotate at a third rate, such that each of the reaction cells sequentially goes through the optical device;

[0015] focusing laser emitted by the excitation light source onto the reaction cells to be detected by the excitation light transmission mirror to excite a reactant in the reaction cells to be detected to generate an optical signal;

[0016] receiving the optical signal by the optical sensor ; and

[0017] calculating a content of each of the pathogenic microorganisms in the sample to be detected according to the optical signal.

[0018] The aforementioned gene detection system based on the microfluidic chip includes the housing, the microfluidic chip, the optical device, and the rotating device. The microfluidic chip is provided with a sample loading cell, a sample dispensing element, and a reaction element. During use, the sample to be detected is loaded in the sample loading cell of the microfluidic chip, and the microfluidic chip is mounted on the rotating device. The rotating device drives the microfluidic chip to rotate for the first time, the sample to be detected enters the arc-shaped channel under the centrifugal action, and sequentially fills the plurality of sample dispensing buffer cells from the inlet end of the arc-shaped channel to the outlet end thereof. Each of the sample dispensing buffer cells has the same volume, the depths of the sample dispensing buffer cells sequentially decrease from the inlet end of the arc-shaped channel to the outlet end thereof, which facilitates the filling of each of the sample dispensing buffer cells by the sample to be detected, thereby ensuring that the volume of the sample to be detected in the sample dispensing buffer cell is equal. Then, the microfluidic chip is driven by the rotating device to rotate for the second time, and the sample to be detected in the sample dispensing buffer cell enters the reaction cells from the capillary tube and reacts with the detection agent loaded in the reaction cells. Thereafter, during the rotation of the microfluidic chip through the rotating device, each of the reaction cells sequentially goes through the optical device, the laser emitted by the excitation light source is focused onto the reaction cells to be detected by the excitation light transmission mirror, and the reactant in the reaction cells to be detected is excited to generate the optical signal. The optical sensor receives the optical signal, so that parameters such as the content of the pathogenic microorganisms in the sample to be detected are calculated and obtained. The aforementioned detection system can detect various pathogenic microorganisms at the same time with a single sample loading, has simple sample processing steps and high detection efficiency. In addition, the amount of the sample loading is easy to control, the volume of the sample to be detected entering each reaction cell is equal, and the detection results are more accurate and the detection process is automated, which meets the application requirements of high efficiency of port health quarantine and quick detection and investigation of large-sample-amount pathogenic microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a schematic view of a detection system in accordance with an embodiment.

[0020] FIG. 2 is a schematic view of the detection system of FIG. 1 in another orientation.

[0021] FIG. 3 is a schematic view of a partial structure of the detection system of FIG. 1.

[0022] FIG. 4 is a schematic view of a partial structure of the detection system of FIG. 1.

[0023] FIG. 5 is a schematic view of a partial structure of the detection system of FIG. 1.

[0024] FIG. 6 is a schematic view of a partial structure of the detection system of FIG. 1.

[0025] FIG. 7 is a diagram of Polymerase chain reaction (PCR) amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Bacillus anthracis having different concentrations using a first group of detection agent.

[0026] FIG. 8 is a diagram of PCR amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Brucella having different concentrations using a second group of detection agent.

[0027] FIG. 9 is a diagram of PCR amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Burkhoderia pseudomallei having different concentrations using a third group of detection agent.

[0028] FIG. 10 is a diagram of PCR amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Francisella tularensis having different concentrations using a fourth group of detection agent.

[0029] FIG. 11 is a diagram of PCR amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Salmonella having different concentrations using a fifth group of detection agent.

[0030] FIG. 12 is a diagram of PCR amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Salmonella typhi having different concentrations using a sixth group of detection agent.

[0031] FIG. 13 is a diagram of PCR amplification curves obtained and a prepared standard curve for fluorescent PCR detection of Shigella having different concentrations using a seventh group of detection agent.

[0032] FIG. 14 is a qPCR amplification curve prepared by changing the fluorescence intensity in each reaction cell with time when detecting a nucleic acid sample of rpoB gene in Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The specific implementation of the present disclosure will be described in detail below with reference to specific embodiments and accompanying drawings.

[0034] Referring to FIGS. 1 and 2, a gene detection system based on a microfluidic chip 01 in accordance with 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 therein, and the microfluidic chip 20 is disposed in the receiving cavity 1001.

[0035] The structure of the microfluidic chip 20 is referring to FIGS. 3 and 4, and the microfluidic chip 20 is substantially circular. The microfluidic chip 20 is provided with a sample loading cell 210, a sample dispensing element 220, and a reaction element 230. One set of the sample loading cell 210, the sample dispensing element 220, and the reaction element 230 form a microfluidic unit 21. The microfluidic chip 20 in accordance with the present embodiment includes four microfluidic units 21 uniformly distributed around a center of a circle. Of course, in other embodiments, the microfluidic chip 20 may also have other shapes, such as rectangular, polygonal, and the like. There may also be one, two, three, five, seven, etc microfluidic units 21 on the microfluidic chip 20.

[0036] Specifically, the sample loading cell 210 is provided with a sample loading hole 2001 in communication with an outside environment. The sample dispensing element 220 includes an arc-shaped channel 221 and a plurality of sample dispensing buffer cells 223, and the arc-shaped channel 221 is in communication with the sample loading cell 210. The plurality of sample dispensing buffer cells 223 are located outside the arc-shaped channel 221 and are sequentially arranged along a circumferential direction of the arc-shaped channel 221. In addition, the sample dispensing buffer cells 223 extend outward from a peripheral edge of the arc-shaped channel 221 along a radial direction of the arc-shaped channel 221. Each of the sample dispensing buffer cells 223 has the same volume, and depths of the sample dispensing buffer cells 223 sequentially decrease from an inlet end of the arc-shaped channel 221 to an outlet end thereof. The reaction element 230 includes a plurality of reaction cells 231 in which detection reagents are loaded, and the reaction cells 231 are in communication with the sample dispensing buffer cell 223 through a capillary tube 240. Specifically, each of the sample dispensing elements 220 includes 16 sample dispensing buffer cells 223 having the same volume, and the number of the reaction cells 231 matches the number of the sample dispensing buffer cells 223. The entire microfluidic chip 20 is provided with 64 sample dispensing buffer cells 223 with the same volume and 64 reaction cells 231, so that high-throughput detection is achieved.

[0037] Specifically, the sample dispensing buffer cell 223 has a rectangular shape and is provided with a chamfer at a bottom thereof. Therefore, after centrifugation, the sample to be detected enters the reaction cell 231 without residue, and the sample actually participating in the reaction is more accurate. Specifically, a depth-to-width ratio of the sample dispensing buffer cell 223 is 1: 1 to 4: 1, the greater the depth-to-width ratio of the inlet end closest to the arc-shaped channel 221 is, the less the depth-to-width ratio of the outlet end closest to the arc-shaped channel 221 is. The depth refers to a distance from an inlet end of the sample dispensing buffer cell 223 to the bottom thereof, and the width refers to a width of an opening of the sample dispensing buffer cell 223. In the present embodiment, the depth-to-width ratio of the inlet end closest to the arc-shaped channel 221 is 4: 1, and the depth-to-width ratio of the outlet end closest to the arc-shaped channel 221 is 1: 1. The sample to be detected can smoothly fill each of the sample dispensing buffer cells 223, so that the sample to be detected in the sample dispensing buffer cells 223 is ensured to be equal in volume.

[0038] Specifically, the sample dispensing element 220 further includes a waste liquid cell 225 disposed at the outlet end of the arc-shaped channel 221 and extending outward along the radial direction of the arc-shaped channel 221. After centrifugation, the sample to be detected sequentially fills the plurality of sample dispensing buffer cells 223 from the inlet end of the arc-shaped channel 221 to the outlet end thereof, and the redundant sample to be detected flows into the waste liquid cell 225, and the sample loading process is convenient and quick.

[0039] Specifically, the reaction element 230 includes a plurality of reaction cells 231 loaded with detection agents, for example, a reaction cell 231 loaded with a bacteria detection agent, a reaction cell 231 loaded with a rickettsia detection agent, a reaction cell 231 loaded with a virus detection agent, a reaction cell 231 loaded with a fungus detection agent, and a reaction cell 231 loaded with a biotoxin detection agent. A distance from a bottom of each of the reaction cells 231 to the edge of the microfluidic chip 20 is equal, for example, 1 mm. Therefore, the propagation distances of the optical paths are equal during detection, and the variation coefficient of optical signal propagation is as small as possible.

[0040] In an embodiment, the reaction element 230 includes reaction cells 231 loaded with the following detection agents, respectively, and each group of the detection agents includes an upstream primer, a downstream primer, and a probe. A first group of detection agent is configured to detect Bacillus anthracis, which includes an upstream primer having a sequence of SEQ ID No. 1, a downstream primer having a sequence of SEQ ID No. 2, and a probe having a sequence of SEQ ID No. 3. The primers and probes described above are designed for the rpoB gene. A second group of detection agent is configured to detect Brucella, which includes an upstream primer having a sequence of SEQ ID No. 4, a downstream primer having a sequence of SEQ ID No. 5, and a probe having a sequence of SEQ ID No. 6. The primers and probes described above are designed for the IS711 gene. A third group of detection agent is configured to detect Burkhoderia pseudomallei, which includes an upstream primer having a sequence of SEQ ID No. 7, a downstream primer having a sequence of SEQ ID No. 8, and a probe having a sequence of SEQ ID No. 9. The primers and probes described above are designed for the fliP gene. A fourth group of detection agent is configured to detect Francisella tularensis, which includes an upstream primer having a sequence of SEQ ID No. 10, a downstream primer having a sequence of SEQ ID No. 11, and a probe having a sequence of SEQ ID No. 12. The primers and probes described above are designed for the tul4 gene. A fifth group of detection agent is configured to detect Salmonella, which includes an upstream primer having a sequence of SEQ ID No. 13, a downstream primer having a sequence of SEQ ID No. 14, and a probe having a sequence of SEQ ID No. 15. The primers and probes described above are designed for the invA gene. A sixth group of detection agent is configured to detect Salmonella typhi, which includes an upstream primer having a sequence of SEQ ID No. 16, a downstream primer having a sequence of SEQ ID No. 17, and a probe having a sequence of SEQ ID No. 18. The primers and probes described above are designed for the staG gene. A seventh group of detection agent is configured to detect Shigella, which includes an upstream primer having a sequence of SEQ ID No. 19, a downstream primer having a sequence of SEQ ID No. 20, and a probe having a sequence of SEQ ID No. 21. The primers and probes described above are designed for the ipaH gene. An eighth group of detection agent is configured to detect Chlamydia psittaci, which includes an upstream primer having a sequence of SEQ ID No. 22, a downstream primer having a sequence of SEQ ID No. 23, and a probe having a sequence of SEQ ID No. 24. The primers and probes described above are designed for the ompA gene. A ninth group of detection agent is configured to detect Rickettsia prowazekii, which includes an upstream primer having a sequence of SEQ ID No. 25, a downstream primer having a sequence of SEQ ID No. 26, and a probe having a sequence of SEQ ID No. 27. The primers and probes described above are designed for the gltA gene. A tenth group of detection agent is configured to detect Ebola virus, which includes an upstream primer having a sequence of SEQ ID No. 28, a downstream primer having a sequence of SEQ ID No. 29, and a probe having a sequence of SEQ ID No. 30. The primers and probes described above are designed for Ebola virus nucleoprotein. An eleventh group of detection agent is configured to detect Hantaan virus, which includes an upstream primer having a sequence of SEQ ID No. 31, a downstream primer having a sequence of SEQ ID No. 32, and a probe having a sequence of SEQ ID No. 33. The primers and probes described above are designed for Hantaan virus nucleoprotein. An twelfth group of detection agent is configured to detect avian influenza virus, which includes an upstream primer having a sequence of SEQ ID No. 34, a downstream primer having a sequence of SEQ ID No. 35, and a probe having a sequence of SEQ ID No. 36. The primers and probes described above are directed to avian influenza virus matrix protein. A thirteenth group of detection agent is configured to detect Variola virus, which includes an upstream primer having a sequence of SEQ ID No. 37, a downstream primer having a sequence of SEQ ID No. 38, and a probe having a sequence of SEQ ID No. 39. The primers and probes described above are designed for the A38R gene. A fourteenth group of detection agent is configured to detect Clostridium botulinum, which includes an upstream primer having a sequence of SEQ ID No. 40, a downstream primer having a sequence of SEQ ID No. 41, and a probe having a sequence of SEQ ID No. 42. The primers and probes described above are designed for the botA gene. A fifteenth group of detection agent is configured to detect Staphylococcus aureus, which includes an upstream primer having a sequence of SEQ ID No. 43, a downstream primer having a sequence of SEQ ID No. 44, and a probe having a sequence of SEQ ID No. 45. The primers and probes described above are designed for the fmhB gene. A sixteenth group of detection agent is configured to detect Arbin, which includes an upstream primer having a sequence of SEQ ID No. 46, a downstream primer having a sequence of SEQ ID No. 47, and a probe having a sequence of SEQ ID No. 48. The primers and probes described above are designed for lectins. Specifically, the reaction element 230 further includes reaction cells 231 loaded with seventeenth group of detection agents, respectively. The seventeenth group of detection agent is configured for positive quality control of bacteria, which includes an upstream primer having a sequence of SEQ ID No. 49, a downstream primer having a sequence of SEQ ID No. 50, and a probe having a sequence of SEQ ID No. 51. The primers and probes described above are designed for the 16S rDNA gene. Through positive quality control, more accurate reaction detection results are obtained.

[0041] Specifically, the 5' end of the probe is provided with a FAM fluorescent group, and the 3' end thereof is provided with a TAMRA fluorescent group. In the detection process, if the detection is carried out on a plurality of pathogenic microorganisms at the same time, the problems that the annealing temperatures are difficult to coordinate during the simultaneous detection and the detection result is inaccurate often occur due to different annealing temperatures of the primers. The research searches for a pathogenic microorganism target gene with specificity, designs and screens the aforementioned detection agents designed aiming at the specific gene or protein respectively. The annealing temperatures of 17 groups of specifically designed detection agent primers are all about 60 °C, the problem of inaccurate detection results caused by different annealing temperatures in simultaneous detection is avoided. 7 bacteria, 2 rickettsias, 4 viruses, 1 fungus, and 2 biotoxins, as well as 1 positive quality control can be simultaneously detected through one-time sample processing, and the detection accuracy is good and the sensitivity is high. Never has the unified amplification condition, realizes large-scale array qPCR, simplifies the realization difficulty of single sample detection based on the conventional amplification tube, improves the qPCR detection throughput and the detection efficiency, and meets the actual requirements of port health quarantine.

[0042] Specifically, one reaction cell 231 is loaded with a group of detection agent. In a group of detection agent, the upstream primer has a concentration of 300 nmol/L to 500 nmol/L, the downstream primer has a concentration of 300 nmol/L to 500 nmol/L, and the probe has a concentration of 200 nmol/L to 400 nmol/L.

[0043] Specifically, the microfluidic chip 20 includes a bottom plate and a top plate. The bottom plate is provided with corresponding grooves of the sample loading cell 210, the sample dispensing buffer cell 223, and the reaction cell 231, and the groove depths are all 2.0 mm. The upstream primer, the downstream primer, and the probe of each group are mixed to prepare a preset solution, and then the preset solution is respectively spotted into the reaction cells 231, and two or more than two reaction cells 231 can contain the same detection agent, so that the results of two or more than two parallel experiments can be obtained in one detection, and the detection accuracy is improved. After the spotting is completed, it is packaged with the top plate and dried or lyophilized at room temperature to obtain the microfluidic chip 20. Preferably, the top plate is a pressure-sensitive film with high transparency, and the pressure-sensitive film with high transparency faces the optical device 30 side during detection. Preferably, the edge of the microfluidic chip 20 is polished to make the variation coefficient of the optical signal propagation as small as possible during detection.

[0044] Specifically, referring to FIG. 5, in the present embodiment, the capillary tube 240 includes a liquid guiding tube 241 and a stopping tube 243, and the capillary tube 240 is subjected to a hydrophobic treatment. The liquid guiding tube 241 is configured to communicate the sample dispensing buffer cell 223 with the reaction cell 231. The stopping tube 243 intersects with the liquid guiding tube 251, and a portion of the liquid guiding tube 241 protrudes outward along a radial direction of the liquid guiding tube 241 to form the stopping tube 243. Specifically, the liquid guiding tube 241 and the stopping tube 243 are combined to form the shape of a cross.

When the sample to be detected enters the reaction cell 231 after the second centrifugation and is subjected to subsequent heating reaction, the solution entering the reaction cell 231 cannot flow back into the sample dispensing buffer cell 223 to cause liquid leakage and cross contamination.

[0045] In an embodiment, the microfluidic chip 20 is further provided with a siphon channel 250 communicating the sample loading cell 210 and the sample dispensing element 220. One end of the siphon channel 250 is connected to the sample loading cell 210, and the other end thereof is connected to the inlet end of the arc-shaped channel 221. The siphon channel 250 is provided with a plurality of curves 251 to prevent liquid from flowing backward. Specifically, the siphon channel 250 is subjected to a hydrophilic treatment, and the siphon channel 250 sucks the liquid in the sample loading cell 210 into the arc-shaped channel 221. Under the action of centrifugal motion, the liquid in the arc-shaped channel 221 sequentially fills the plurality of sample dispensing buffer cells 223, and redundant sample to be detected flows into the waste liquid cell 225.

[0046] In an embodiment, the microfluidic chip 20 is further provided with an exhaust pipe 260 configured to conduct air in the sample loading cell 210 and the sample dispensing element 220. One end of the exhaust pipe 260 is connected to the sample loading cell 210, and the other end of the exhaust pipe 260 is connected to the outlet end of the arc-shaped channel 221. By providing the exhaust pipe 260, the gas pressure in the sample loading cell 210 and the arc-shaped channel 221 is balanced to facilitate the liquid in the arc-shaped channel 221 to sequentially fill the plurality of sample dispensing buffer cells 223. Specifically, part of the exhaust pipe 260 protrudes outward along a radial direction of the exhaust pipe 260 to form an exhaust cavity 261, and the exhaust cavity 261 is provided with an exhaust hole 2003 in communication with an outside environment. After the sample to be detected is filled in the plurality of sample dispensing buffer cells 223, the extruded gas enters the exhaust cavity 261 through the exhaust pipe 260 and is discharged through the exhaust hole 2003. The volume at the exhaust cavity 261 is large to prevent the liquid from being splashed out.

[0047] In an embodiment, the microfluidic chip 20 is provided with a heat conduction channel 270, and the heat conduction channel 270 extends through the microfluidic chip 20. During heating, the airflow on both sides of the microfluidic chip 20 is conducted through the heat conduction channel 270, so that the microfluidic chip 20 is uniformly heated.

[0048] Specifically, the microfluidic chip 20 has a circular shape, the sample loading cell 210, the sample dispensing element 220, and the reaction element 300 are sequentially distributed outward along the radial direction of the microfluidic chip 20, and the arc-shaped channel 210 is arranged concentrically with the microfluidic chip 20.

[0049] Specifically, referring to FIG. 6, the optical device 30 includes an excitation light source 310, an excitation light transmission mirror 320, an emergent light lens 330, a filter 340, and an optical sensor 350. The excitation light source 310 is configured to emit laser, and the excitation light transmission mirror 320 is configured to focus and irradiate the laser onto the reaction cells 231 to be detected so as to excite a reactant in the reaction cells 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. The reactant in the reaction cell 231 generate the optical signal under the irradiation of the excitation light, and the emergent light lens 330 is configured to focus the optical signal generated by the reactant in the reaction cell 231. The filter 340 is configured to filter the focused optical signal before transmitting to the optical sensor 350, and the optical sensor 350 receives and records the signal intensity of each reaction cell 231. The optical sensor 350 is, for example, a photomultiplier tube or a photodiode. Of course, in other embodiments, the emergent light lens 330 and the filter 340 may be omitted.

[0050] In an embodiment, the laser emitted from the excitation light source 310 is irradiated on the reaction cell 231 to be detected, the laser is perpendicular to a plane on which the microfluidic chip 20 is located. The excitation light source 310 is facing the microfluidic chip 20, and the emitted laser is irradiated on the reaction cell 231. Specifically, the optical sensor 350 is mounted on the housing 10 and is parallel to the plane on which the microfluidic chip 20 is located. During the rotation of the microfluidic chip 20, optical signals emitted from each reaction cell 231 are received. Specifically, during the detection, 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 to be detected, the emergent light lens 330, the filter 340, and the optical sensor 350 are in a straight line. In addition, the emergent light lens 330, the filter 340, and the optical sensor 350 are disposed adjacent to the edge of the microfluidic chip 20. The straight line formed by the excitation light source 310, the excitation light transmission mirror 320, and the reaction cell 231 to be detected intersects with the straight line formed by the reaction cell 231 to be detected, the emergent light lens 330, the filter 340, and the optical sensor 350 at the reaction cell 231 to be detected, and the two straight lines are perpendicular to each other.

[0051] Specifically, the rotating device 40 is configured to drive the microfluidic chip 20 to rotate, so that each of the reaction cells 231 sequentially goes through the optical device 30, thereby achieving the automation of the detection process. The rotating device 40 is, for example, a rotary electric machine.

[0052] Referring to FIG. 1 again, in an embodiment, the detection system 01 further includes a heater 50 and a cooler 60, which are both located in the receiving cavity 1001. The heater 50 includes a heat source 51 and a heat sink 53, and the heat source 51 is configured to provide heat energy. The heat sink 53 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 diffuse into the receiving cavity 1001, so that the microfluidic chip 20 is uniformly heated. The cooler 60 is configured to cool the microfluidic chip 20. During the reaction, depending on the progress, the temperature needs to be quickly switched so that the sample to be detected and the detection agent can be smoothly reacted in the reaction cell 231. Specifically, the cooler 60 is disposed directly below the microfluidic chip 20.

[0053] The heater 50 and the cooler 60 provided above achieve synchronous and rapid temperature control of the high-throughput microfluidic chip by using a non-contact balanced temperature control technology. Compared with the conventional heating mode based on thermoelectric semiconductor (Peltier), the temperature control technology has the following advantages: a) the temperature control cost is reduced, the conventional contact type thermoelectric semiconductor module is no longer relied on, and the temperature control technology is more suitable for the integral temperature control of a small microfluidic chip; b) the temperature is increased and decreased quickly, and the uniformity of the heat transfer temperature of the rotary air is better; and c) the expansion compatibility is strong, the temperature control and the optimization test can be well carried out on the microfluidic chips with different shapes and different structures, the size limitation of the conventional contact type temperature control module is eliminated, and the edge effect is avoided.

[0054] In an embodiment, the housing 10 is provided with a gas passage switch 1003. The housing 10 is hermetically sealed, and the gas passage switch 1003 is opened or closed to accelerate the gas convection between the receiving cavity 1001 and the outside.

[0055] Specifically, the microfluidic chip 20 is detachably disposed on the rotating device 40. The detection system 01 further includes a locking mechanism 70 for locking the microfluidic chip , through which the microfluidic chip 20 is prevented from deviating from the running track during the centrifugation.

[0056] The aforementioned detection system 01 can detect various pathogenic microorganisms at the same time with a single sample loading, has simple sample processing steps and high detection efficiency. In addition, the amount of the sample loading is easy to control, the volume of the sample to be detected entering each reaction cell 231 is equal, and the detection results are more accurate and the detection process is automated, which meets the application requirements of high efficiency of port health quarantine and quick detection and investigation of large-sample-amountpathogenicmicroorganisms.

[0057] A method of detecting content of pathogenic microorganisms in accordance with an embodiment includes the following steps S110 to S150.

[0058] At step S110, a sample to be detected is loaded in a sample loading cell of a detection system shown in FIG. 1.

[0059] Specifically, the sample to be detected may be a serum, powder, and the like. The sample to be detected is formulated as a solution and loaded to the sample loading cell 210 through sample loading hole 2001.

[0060] At step S120, a microfluidic chip is driven to centrifugally rotate at a first rate by a rotating device, so that the sample to be detected in the sample loading cell sequentially enters a plurality of sample dispensing buffer cells through an arc-shaped channel.

[0061] Specifically, the first rate ranges from 800 rpm to 1000 rpm. The sample to be detected enters the arc-shaped channel 210 by centrifugation, and sequentially fills the plurality of sample dispensing buffer cells 223 from an inlet end of the arc-shaped channel 221 to an outlet end thereof, and the redundant sample to be detected flows into a waste liquid cell 225.

[0062] At step S130, the microfluidic chip is driven to centrifugally rotate at a second rate by the rotating device, so that the sample to be detected in sample dispensing buffer cells enters a reaction cell through a capillary tube.

[0063] Specifically, the second rate ranges from 2500 rpm to 3000 rpm. Under the action of a large centrifugal rate, the sample to be detected in the sample dispensing buffer cell 223 enters the reaction cell 231 through the capillary tube 240 and reacts with detection agents previously stored in the reaction cell 231.

[0064] At step S140, the microfluidic chip is driven to centrifugally rotate at a third rate by the rotating device, so that each of the reaction cells sequentially goes through an optical device.

Laser emitted by an excitation light source is focused onto the reaction cell to be detected by an excitation light transmission mirror to excite a reactant in the reaction cell to be detected to generate an optical signal. The optical sensor receives the optical signal.

[0065] Specifically, the third rate ranges from 200 rpm to 600 rpm. The microfluidic chip 20 rotates at a lower rate, and the reaction cell 231 sequentially goes through the optical device 30, and the corresponding reaction optical signal is detected and obtained by the optical device 30.

[0066] At step S150, a content of each of the pathogenic microorganisms in the sample to be detected is calculated according to the optical signal.

[0067] For example, the optical sensor records the fluorescence signal intensity values of all the reaction cells 231 in the reaction process, draws corresponding amplification curves, and analyzes the reaction results to obtain the content of each of the pathogenic microorganisms in the sample to be detected.

[0068] The aforementioned detection method can directly detect unprocessed complex samples, such as serum, powder, and the like, and has the capability of synchronously and quickly detecting various pathogens by single reaction, shortens the response time of port health quarantine and improves the detection efficiency. The aforementioned detection method is highly automated, avoids potential biological safety threat, can quickly achieve high-throughput distribution of samples of the same biological sample or clinical sample by a centrifugal microfluidic chip technology, reduces reagent consumption and time consumption, and has significant cost-effectiveness.

[0069] The specific detection example 1 is described in detail as follows.

[0070] The microfluidic chip high-throughput gene detection system 01 was shown in FIG. 1, and a reaction element 230 included 64 reaction cells 231, which were labeled as No. 1 to No. 64, respectively. The reaction cells No. 1 to No. 3 were loaded with the first group of detection agents with high, medium, and low concentrations, respectively. The reaction cells No. 4 to No. 51 were loaded with the second group of detection agents to the sixteenth group of detection agents with high, medium, and low concentrations, respectively. The reaction cell No. 52 was loaded with the seventeenth group of detection agent (positive control). The reaction cells No. 53 to No. 64 were blank controls. The detection agents with high concentrations in the reaction cells No. 1 to No. 51 included an upstream primer having a concentration of 500 nmol/L, a downstream primer having a concentration of 500 nmol/L, and a probe having a concentration of 400 nmol/L. The detection agents with medium concentrations included an upstream primer having a concentration of 400 nmol/L, a downstream primer having a concentration of 400 nmol/L, and a probe having a concentration of 300 nmol/L. The detection agents with low concentrations included an upstream primer having a concentration of 300 nmol/L, a downstream primer having a concentration of 300 nmol/L, and a probe having a concentration of 200 nmol/L. The seventeenth group of detection agent included an upstream primer having a concentration of 400 nmol/L, a downstream primer having a concentration of 400 nmol/L, and a probe having a concentration of 300 nmol/L. A preliminary experiment was performed first, in which the first group of detection agent was used to perform fluorescent polymerase chain reaction (PCR) detection of Bacillus anthracis having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 7. The second group of detection agent was used to perform fluorescent PCR detection of Brucella having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 8. The third group of detection agent was used to perform fluorescent PCR detection of Burkhoderia pseudomallei having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 9. The fourth group of detection agent was used to perform fluorescent PCR detection of Francisella tularensis having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 10. The fifth group of detection agent was used to perform fluorescent PCR detection of Salmonella having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 11. The sixth group of detection agent was used to perform fluorescent PCR detection of Salmonella typhi having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 12. The seventh group of detection agent was used to perform fluorescent PCR detection of Shigella having different concentrations, thereby obtaining PCR amplification curves and preparing a standard curve shown in FIG. 13. Each group of detection agent included an upstream primer having a concentration of 400 nmol/L, a downstream primer having a concentration of 400 nmol/L, and a probe having a concentration of 300 nmol/L. As can be seen from FIGS. 7 to 13, the standard curve prepared by each group of detection agents has good linearity, which indicates that the designed primers have good specificity and high sensitivity, and the annealing temperature of the primer of each group of detection agents is about 60 °C, so that the problem of inaccurate detection results caused by different annealing temperatures in simultaneous detection is avoided, and each group of detection agents can be used for detecting on one microfluidic chip 20.

[0071] Then, a sample to be detected (a nucleic acid sample of rpoB gene) was loaded into the sample loading cell 210 through sample loading hole 2001. The microfluidic chip 20 after sample loading was placed on the rotating device 40 and locked by the locking mechanism 70. The detection system 01 was started, the microfluidic chip 20 was driven to be centrifuged at 800 rpm for 1 minute by the rotating device 40. The sample to be detected entered the arc-shaped channel 210 by the centrifugation, and sequentially filled the plurality of sample dispensing buffer cells 223 from the inlet end of the arc-shaped channel 221 to the outlet end thereof. Each of the sample dispensing buffer cells 223 had the same volume, the depths of the sample dispensing buffer cells 223 sequentially decreased from the inlet end of the arc-shaped channel 210 to the outlet end thereof, which facilitates the filling of each of the sample dispensing buffer cells 223 by the sample to be detected, thereby ensuring that the volume of the sample to be detected in the sample dispensing buffer cell 223 is equal. Then, the microfluidic chip 20 was driven to be centrifuged at 2500 rpm for 2 minutes by the rotating device 40, so that the sample to be detected in each of the sample dispensing buffer cells 223 entered the reaction cell 231 through the capillary tube 240 and reacted with the detection agent previously stored in the reaction cell 231. The centrifugal rate was then reduced to 400 rpm. The heater was started for heating, and the heat source 51 started to increase the temperature to heat the air in the receiving cavity 1001, and at the same time, the heat sink 53 started to work and caused the internal circulation airflow to stir the hot air, so that the microfluidic chip 20 was uniformly heated. After reaching the target temperature of 95 °C, the heat source 51 would reduce the power to maintain the denaturation temperature required for the PCR of the microfluidic chip 20. Thereafter, the gas passage switch 1003 was opened, and the heat source 51 stopped working. The cooler 60 started to work, external cold air was introduced into the system to replace original hot air to finish cooling. After reaching the target temperature of 60 °C, the gas passage switch 1003 was closed, meanwhile, the cooler 60 stopped working, and the extension time required by the PCR of the microfluidic chip 20 was maintained. Thus, a round of PCR cycle was completed. The optical device 30 completed the fluorescence detection of the reaction cell 231 within the extension time for performing the PCR of the microfluidic chip 20. When the temperature in the receiving cavity 1001 reached 60 °C, the excitation light source 310 started to work, meanwhile, the reaction cell 231 of the microfluidic chip 20 would sequentially pass through the light path of the excitation light at a rotation speed of 400 rpm. The excitation light was focused in the reaction cell 231 under the action of the excitation light transmission mirror 320, and the fluorescence signal generated by the hydrolysis probe for the qPCR reaction of the detection agent (such as the primer and the probe) in the reaction cell 231 was excited. The fluorescence signal sequentially passed through the emergent light lens 330 and the filter 340, and was finally received by the optical sensor 350. The optical sensor 350 received and recorded the current cycle signal intensity. After 40 cycles, the system would record the fluorescence signal intensity values of all the reaction cells 231 for 40 cycles, and drawn the corresponding qPCR amplification curves, with the results shown in FIG. 14. The four reaction cells Nos. 1 to 3 and No. 52 have qPCR amplification curves, and the reaction cells with other numbers have no qPCR amplification curves. The detection system 01 can obtain the optical signal of each reaction cell 231 and simultaneously detect various pathogenic microorganisms, and has simple sample processing steps and high detection efficiency.

[0072] Although the respective exemplary embodiments have been described one by one, it shall be appreciated that the respective exemplary embodiments will not be isolated. Those skilled in the art can apparently appreciate upon reading the disclosure of this application that the respective technical features involved in the respective exemplary embodiments can be combined arbitrarily between the respective exemplary embodiments as long as they have no collision with each other. Of course, the respective technical features mentioned in the same exemplary embodiment can also be combined arbitrarily as long as they have no collision with each other.

[0073] The foregoing descriptions are merely specific exemplary embodiments of the present disclosure, but are not intended to limit the protection scope of the present disclosure. Any variation or replacement readily figured out by a person skilled in the art within the technical scope disclosed in the present disclosure shall all fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the appended claims.

Claims

What is claimed is: 1. A gene detection system based on a microfluidic chip, comprising: a housing provided with a receiving cavity therein; a microfluidic chip disposed in the receiving cavity, the microfluidic chip being provided with a sample loading cell, a sample dispensing element, and a reaction element, the sample loading cell being configured to load a sample to be detected, the sample dispensing element comprising an arc-shaped channel and a plurality of sample dispensing buffer cells, the arc-shaped channel being in communication with the sample loading cell, the plurality of sample dispensing buffer cells being located outside the arc-shaped channel and being sequentially arranged along a circumferential direction of the arc-shaped channel, the sample dispensing buffer cell extending outward from a peripheral edge of the arc-shaped channel along a radial direction of the arc-shaped channel, each of the sample dispensing buffer cells having the same volume, depths of the sample dispensing buffer cells sequentially decreasing from an inlet end of the arc-shaped channel to an outlet end thereof, the reaction element comprising a reaction cell loaded with a bacteria detection agent, a reaction cell loaded with a rickettsia detection agent, a reaction cell loaded with a virus detection agent, a reaction cell loaded with a fungus detection agent, and a reaction cell loaded with a biotoxin detection agent, the reaction cells being in communication with the sample dispensing buffer cell through a capillary tube; an optical device comprising an excitation light source, an excitation light transmission mirror, and an optical sensor, the excitation light source being configured to emit laser, the excitation light transmission mirror being configured to focus and irradiate the laser onto the reaction cells to be detected so as to excite a reactant in the reaction cells to be detected to generate an optical signal, and the optical sensor being configured to receive the optical signal; and a rotating device configured to drive the microfluidic chip to rotate so that each of the reaction cells sequentially goes through the optical device.
2. The detection system of claim 1, wherein the reaction element comprises reaction cells loaded with the following detection agents respectively, each group of the detection agents comprises an upstream primer, a downstream primer, and a probe; a first group of detection agent configured to detect Bacillus anthracis, comprising an upstream primer having a sequence of SEQ ID No. 1, a downstream primer having a sequence of SEQ ID No.

2, and a probe having a sequence of SEQ ID No. 3;

a second group of detection agent configured to detect Brucella, comprising an upstream primer

having a sequence of SEQ ID No. 4, a downstream primer having a sequence of SEQ ID No. 5, and

a probe having a sequence of SEQ ID No. 6;

a third group of detection agent configured to detect Burkhoderia pseudomallei, comprising an

upstream primer having a sequence of SEQ ID No. 7, a downstream primer having a sequence of

SEQ ID No. 8, and a probe having a sequence of SEQ ID No. 9;

a fourth group of detection agent configured to detect Francisella tularensis, comprising an upstream

primer having a sequence of SEQ ID No. 10, a downstream primer having a sequence of SEQ ID

No. 11, and a probe having a sequence of SEQ ID No. 12;

a fifth group of detection agent configured to detect Salmonella, comprising an upstream primer

having a sequence of SEQ ID No. 13, a downstream primer having a sequence of SEQ ID No. 14,

and a probe having a sequence of SEQ ID No. 15;

a sixth group of detection agent configured to detect Salmonella typhi, comprising an upstream

primer having a sequence of SEQ ID No. 16, a downstream primer having a sequence of SEQ ID

No. 17, and a probe having a sequence of SEQ ID No. 18;

a seventh group of detection agent configured to detect Shigella, comprising an upstream primer

having a sequence of SEQ ID No. 19, a downstream primer having a sequence of SEQ ID No. 20,

and a probe having a sequence of SEQ ID No. 21;

an eighth group of detection agent configured to detect Chlamydia psittaci, comprising an upstream

primer having a sequence of SEQ ID No. 22, a downstream primer having a sequence of SEQ ID

No. 23, and a probe having a sequence of SEQ ID No. 24;

a ninth group of detection agent configured to detect Rickettsia prowazekii, comprising an upstream

primer having a sequence of SEQ ID No. 25, a downstream primer having a sequence of SEQ ID

No. 26, and a probe having a sequence of SEQ ID No. 27; a tenth group of detection agent configured to detect Ebola virus, comprising an upstream primer having a sequence of SEQ ID No. 28, a downstream primer having a sequence of SEQ ID No. 29, and a probe having a sequence of SEQ ID No. 30; an eleventh group of detection agent configured to detect Hantaan virus, comprising an upstream primer having a sequence of SEQ ID No. 31, a downstream primer having a sequence of SEQ ID

No. 32, and a probe having a sequence of SEQ ID No. 33;

an twelfth group of detection agent configured to detect avian influenza virus, comprising an

upstream primer having a sequence of SEQ ID No. 34, a downstream primer having a sequence of

SEQ ID No. 35, and a probe having a sequence of SEQ ID No. 36;

a thirteenth group of detection agent configured to detect Variola virus, comprising an upstream

primer having a sequence of SEQ ID No. 37, a downstream primer having a sequence of SEQ ID

No. 38, and a probe having a sequence of SEQ ID No. 39;

a fourteenth group of detection agent configured to detect Clostridium botulinum, comprising an

upstream primer having a sequence of SEQ ID No. 40, a downstream primer having a sequence of

SEQ ID No. 41, and a probe having a sequence of SEQ ID No. 42;

a fifteenth group of detection agent configured to detect Staphylococcus aureus, comprising an

upstream primer having a sequence of SEQ ID No. 43, a downstream primer having a sequence of

SEQ ID No. 44, and a probe having a sequence of SEQ ID No. 45; and

a sixteenth group of detection agent configured to detect Arbin, comprising an upstream primer

having a sequence of SEQ ID No. 46, a downstream primer having a sequence of SEQ ID No. 47,

and a probe having a sequence of SEQ ID No. 48.
3. The detection system of claim 1, wherein in the detection agent, the upstream primer has a

concentration of 300 nmol/L to 500 nmol/L, the downstream primer has a concentration of 300

nmol/L to 500 nmol/L, and the probe has a concentration of 200 nmol/L to 400 nmol/L.
4. The detection system of claim 1, wherein the optical device further comprises an emergent light

lens and a filter, the emergent light lens is configured to focus the optical signal generated by the reactant in the reaction cells, and the filter is configured to filter the focused optical signal before transmitting to the optical sensor.
5. The detection system of claim 1, wherein the laser emitted from the excitation light source is

irradiated on the reaction cells to be detected, the laser is perpendicular to a plane on which the

microfluidic chip is located, and the optical sensor is mounted on the housing and is parallel to the

plane on which the microfluidic chip is located.
6. The detection system of claim 1, wherein the microfluidic chip is further provided with:

a waste liquid cell disposed at the outlet end of the arc-shaped channel and extending outward along

the radial direction of the arc-shaped channel;

a siphon channel communicating the sample loading cell and the sample dispensing element, one

end of the siphon channel being connected to the sample loading cell, the other end of the siphon

channel being connected to the inlet end of the arc-shaped channel, and the siphon channel is

provided with a plurality of curves; and

an exhaust pipe configured to conduct air in the sample loading cell and the sample dispensing

element, one end of the exhaust pipe being connected to the sample loading cell, the other end of the

exhaust pipe being connected to the outlet end of the arc-shaped channel, wherein part of the

exhaust pipe protrudes outward along a radial direction of the exhaust pipe to form an exhaust

cavity, and the exhaust cavity is provided with an exhaust hole in communication with an outside

environment.
7. The detection system of claim 1, wherein the sample dispensing buffer cell has a rectangular

shape and is provided with a chamfer at a bottom thereof, a depth-to-width ratio of the sample

dispensing buffer cell is 1: 1 to 4: 1.
8. The detection system of claim 1, further comprising:

a heater located in the receiving cavity, the heater comprising a heat source and a heat sink, the heat source being configured to provide heat energy, and the heat sink being disposed around the heat source; and a cooler located in the receiving cavity and configured to cool the microfluidic chip.
9. A method of detecting content of pathogenic microorganisms, comprising:

loading a sample to be detected in the sample loading cell of the detection system of any one of

claims 1 to 8;

driving, by the rotating device, the microfluidic chip to centrifugally rotate at a first rate, such that

the sample to be detected in the sample loading cell sequentially enters the plurality of sample

dispensing buffer cells through the arc-shaped channel;

driving, by the rotating device, the microfluidic chip to centrifugally rotate at a second rate, such

that the sample to be detected in sample dispensing buffer cells enters the reaction cells through the

capillary tube;

driving, by the rotating device, the microfluidic chip to centrifugally rotate at a third rate, such that

each of the reaction cells sequentially goes through the optical device;

focusing laser emitted by the excitation light source onto the reaction cells to be detected by the

excitation light transmission mirror to excite a reactant in the reaction cells to be detected to

generate an optical signal;

receiving the optical signal by the optical sensor; and

calculating a content of each of the pathogenic microorganisms in the sample to be detected

according to the optical signal.
10. The method of claim 9, wherein the first rate ranges from 800 rpm to 1000 rpm, the second rate

ranges from 2500 rpm to 3000 rpm, and the third rate ranges from 200 rpm to 600 rpm.