CN114196725B - Method and system for identifying microorganism - Google Patents
Method and system for identifying microorganism Download PDFInfo
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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Abstract
The present invention relates to a method and a system for identifying microorganisms. The method comprises the following steps: introducing bacterial liquid containing microorganisms to be detected into a first detection channel of a preliminary identification device, applying voltage to the side wall of the first detection channel, recording a voltage signal, judging whether the microorganisms to be detected are agglomerated or not based on the change of the voltage signal, and collecting the microorganisms which are not agglomerated; and introducing the microorganisms which are not agglomerated into a second detection channel of the Raman identification device, emitting laser to the second detection channel, recording Raman signals, drawing a spectrogram based on the Raman signals, comparing the spectrogram with the spectrogram of the known microorganisms, and determining the type of the microorganisms to be detected. The method can sort and identify single microorganisms in the bacterial liquid so as to screen target microorganisms for targeted culture, has the advantages of improving culture efficiency, realizing specific culture, reducing cost and the like, and provides a new mode for microorganism research and future diagnosis and treatment.
Description
Technical Field
The invention relates to the field of microorganism identification, in particular to a method and a system for identifying microorganisms.
Background
Microorganisms are national strategic resources, the number of the microorganisms in human body colonisation reaches thousands of species, the number of the microorganisms reaches millions, the research on the health correlation of the microorganisms and the diagnosis and treatment application thereof has profound significance, but the characteristics of a plurality of microorganisms are still unknown, and the research is needed. However, the traditional microorganism research method mainly comprises the steps of identifying microorganisms after culturing, wherein the survival conditions of the microorganisms are complex, and nearly 80% of bacteria are not successfully cultured under the conventional culturing means and cannot enter the subsequent microorganism identification link so as to research the influence mechanism.
Thus, a new microorganism identification and culture technique is needed.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a method and a system for identifying microorganisms, and the method can realize real-time in-situ identification, high-throughput intelligent automatic screening and identification of microorganisms in a sample so as to obtain target microorganisms.
The present invention has been completed based on the following work of the inventors:
in the existing culture technology, the primary technology adopts a culture-identification technology route, and utilizes a solid culture medium to separate monoclonal and then carry out biochemical identification, so that the requirement is low, the time and the labor are wasted, and unknown bacteria cannot be completely identified. In order to improve the first generation technology, researchers utilize mass spectrometry to screen and then selectively sequence monoclonal, so as to form a culture-screening-identification culture set technical route, the separation efficiency is improved to a certain extent, the bottleneck that the identification of the non-cultured bacteria cannot be realized in the first generation is not broken through, a large amount of labor time is required, the automation degree is low, and the anaerobic bacteria automatic culture is difficult to realize.
Based on the method, the inventor provides an identification-screening-culture technology route, the inventor can obtain single microorganisms in bacterial liquid through a microfluidic channel, then carries out real-time in-situ identification, high-throughput intelligent automatic identification and screening on the single microorganisms by adopting Raman spectrum, and cultures the target microorganisms according to the identification result targeting matching optimal culture conditions after screening the target microorganisms.
In one aspect of the invention, the invention provides a method of identifying a microorganism. According to an embodiment of the invention, the method comprises: (1) Introducing bacterial liquid containing microorganisms to be detected into a first detection channel of a preliminary identification device, applying voltage to the side wall of the first detection channel, recording a voltage signal, judging whether the microorganisms to be detected are agglomerated or not based on the change of the voltage signal, and collecting the microorganisms which are not agglomerated; (2) And introducing the microorganisms which are not agglomerated into a second detection channel of a Raman identification device, emitting laser to the second detection channel, recording a Raman signal, drawing a spectrogram based on the Raman signal, comparing the spectrogram with a spectrogram of a known microorganism, and determining the type of the microorganism to be detected.
By adopting the method, whether the microorganisms to be detected are agglomerated or not is judged based on the voltage signal change of the microorganisms, then real-time in-situ and high-throughput intelligent automatic identification is carried out on the microorganisms which are not agglomerated, the identification result and a spectrogram of the known microorganisms are analyzed, the types of the microorganisms are determined so as to screen out target microorganisms, and the target microorganisms can be directly subjected to targeted culture in the follow-up process so as to improve the culture efficiency and realize specific culture.
According to an embodiment of the present invention, the method may further comprise at least one of the following additional technical features:
according to an embodiment of the present invention, the bacterial liquid is subjected to dilution treatment in advance before the step (1) is performed.
According to an embodiment of the present invention, after step (2) is performed, the determined type of microorganism to be tested is cultured.
According to an embodiment of the invention, the applied voltage is applied by means of an electrode pair on the side wall of the first detection channel.
According to an embodiment of the present invention, whether the microorganism to be tested is agglomerated is determined by: according to the formulaCalculating the number of microorganisms, wherein I: a current; t: time; Δε: a change in permittivity caused by a single cell; a: electrode equivalent area; v (V) 1 : voltage value.
According to an embodiment of the invention, the number of microorganisms greater than 1 is an indication of agglomeration of the microorganisms to be tested.
According to an embodiment of the present invention, the number of microorganisms being 1 or less is an indication that the microorganisms to be tested are not agglomerated.
According to an embodiment of the present invention, in step (1), further comprising: and acquiring image information of the microorganisms to be detected in the first detection channel, and primarily identifying the types of the microorganisms to be detected based on the image information.
According to the embodiment of the invention, the outlet of the first detection channel is respectively communicated with the inlet of the target microorganism channel and the inlet of the waste liquid channel, and the inner diameter of the inlet of the target microorganism channel is smaller than that of the inlet of the waste liquid channel; the outlet of the target microorganism channel is communicated with the inlet of the second detection channel.
According to an embodiment of the invention, the method further comprises: applying pressure to the outlet of the first detection channel to cause the non-agglomerated microorganisms to pass through the target microorganism channel into the second detection channel.
According to an embodiment of the present invention, the raman authentication device includes a plurality of second detection channels, and the laser wavelength emitted to each of the second detection channels is different, and the second detection channels have a serpentine shape.
According to an embodiment of the invention, a voltage is applied at the inlet of the second detection channel to control the flow rate and/or time of the non-agglomerated microorganisms into the second detection channel.
In yet another aspect of the invention, the invention provides a system for performing the above method of identifying microorganisms. The system comprises: the primary identification device comprises a first detection channel, and an electrode pair is arranged on the first detection channel; a raman authentication device comprising a second detection channel and a laser emitting component adapted to emit laser light into the second detection channel; the power supply device is connected with the electrode pair on the first detection channel; an electrical signal collection device adapted to collect electrical signals of the electrode pairs; an optical signal collection device adapted to collect a laser signal of a specific position irradiated by the laser emitting part; and the analysis device is respectively connected with the electric signal collection device and the optical signal collection device.
By adopting the system, the bacterial liquid of the microorganism to be detected is guided into the preliminary identification device, the electric signal collection device receives the electric signal information detected in the preliminary identification device and sends the electric signal information to the analysis device for analysis, the analysis device judges whether the microorganism passing through the first detection channel is agglomerated or not, then the microorganism which is not agglomerated is guided into the Raman identification device, meanwhile, the optical signal collection device receives the spectral information detected in the Raman identification device and sends the spectral information to the analysis device for analysis, so as to determine the type of the microorganism, the target microorganism can be screened subsequently, and the target microorganism can be directly subjected to targeted culture according to the identification result, so that the culture efficiency is improved, the specific culture is realized, the cost is reduced, and the like, and a new mode can be provided for microorganism research and future diagnosis and treatment.
According to an embodiment of the invention, the first detection channel is further provided with an image recognition means, preferably a CCD image sensor.
According to the embodiment of the invention, the outlet of the first detection channel is respectively communicated with the inlet of the target microorganism channel and the inlet of the waste liquid channel, and the inner diameter of the inlet of the target microorganism channel is smaller than that of the inlet of the waste liquid channel; the outlet of the target microorganism channel is communicated with the inlet of the second detection channel.
According to an embodiment of the invention, the outlet of the first detection channel is provided with a pressurizing device.
According to an embodiment of the present invention, an electrode pair is disposed on an inlet of the second detection channel, and the electrode pair is connected to the power supply device.
According to an embodiment of the invention, the system further comprises: and the culture device is connected with the Raman identification device.
According to an embodiment of the present invention, the raman authentication device includes a plurality of the second detection channels, the second detection channels are in a serpentine shape, the bottom is a concave surface, and a focal position of the concave surface coincides with a focal point of the laser emitting component.
According to an embodiment of the invention, the system further comprises: and the control part is respectively connected with the preliminary identification device, the Raman identification device, the power supply device, the electric signal collection device, the optical signal collection device, the analysis device, the image identification part, the pressurizing device and the culture device.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a flow chart of a method of identifying microorganisms in one embodiment of the present invention;
FIG. 2 is a schematic diagram showing the measurement of the movement speed of microorganisms by electrode pairs according to an embodiment of the present invention;
FIG. 3 is a schematic view showing a mounting structure between a first detection channel and an electrode pair in an embodiment of the present invention;
FIG. 4 is a schematic flow chart of a method for identifying microorganisms in one embodiment of the present invention;
FIG. 5 is a schematic diagram of a system for identifying microorganisms in one embodiment of the present invention;
FIG. 6 is a schematic view of a pressurizing apparatus for sorting microorganisms according to one embodiment of the present invention;
FIG. 7 is a schematic diagram showing a concave design at the bottom of the second detection channel according to an embodiment of the present invention;
FIG. 8 is a schematic diagram of the placement of electrode pairs in an embodiment of the invention;
FIG. 9 is a schematic diagram of a Raman authentication device according to an embodiment of the present invention;
FIG. 10 is a schematic diagram showing a Raman spectrum of a microorganism and a Raman spectrum of a marker detected by a Raman identifying apparatus according to an embodiment of the present invention;
FIG. 11 is a graph showing Raman spectrum data of intestinal core microorganisms detected by a Raman identification device according to an embodiment of the present invention.
Reference numerals:
000. a pretreatment device; 100. a preliminary authentication device; 110. a first detection channel; 120. an image recognition section; 200. a Raman identification device; 210. a second detection channel; 220. an optical path coupling device; 230. a laser emitting part; 300. an optical signal collection device; 310. a raman spectrum signal processor; 400. an analysis device; 500. a control part; 600. a culture device; 700. a waste liquid recovery device; 800. and a pressurizing device.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The present invention provides a method for identifying microorganisms and a system thereof, which will be described in detail below, respectively.
Method for identifying microorganisms
In one aspect of the invention, the invention provides a method of identifying a microorganism. According to an embodiment of the present invention, as shown in fig. 1, the method is performed as follows:
s100, preliminary sorting: and (3) introducing bacterial liquid containing microorganisms to be detected into a first detection channel of the preliminary identification device, applying voltage to the side wall of the first detection channel, recording a voltage signal, judging whether the microorganisms to be detected are agglomerated or not based on the change of the voltage signal, and collecting the microorganisms which are not agglomerated.
According to the embodiment of the invention, the bacterial liquid of the microorganism to be detected is guided into the first detection channel, and meanwhile, voltage is applied to obtain a voltage signal of the microorganism, and whether the microorganism to be detected is agglomerated or not is judged according to the change of the voltage signal so as to collect the microorganism which is not agglomerated.
In some embodiments, the applying a voltage is performed by a pair of electrodes on the side wall of the first detection channel. Thereby, by acquiring the capacitance change between the electrode pairs, a voltage signal change is obtained; and through the arrangement of the electrode pairs, the voltage of the electrode pairs in the first detection channel can be controlled so as to avoid the contact between microorganisms and the inner wall of the first detection channel and realize the adsorption and desorption of the microorganisms. Therefore, the electrode pair can be used as a sensor and a controller, can detect microorganisms through capacitance change and can control the flow speed and/or time of the microorganisms through microorganism adsorption and desorption, so that the full-flow low-cost dynamic tracking and regulation of the microorganisms are realized.
In some embodiments, as shown in fig. 2, the electrode pairs are arranged in a plurality of pairs. By multiple (e.g. k) pairs of electrodesIs the total time t of (2) 2 And the center-to-center distance d between the electrode pairs 2 Can realize the rapid measurement of the average moving speed v of the microorganism, namely v=kd 2 /t 2 。
In some embodiments, the voltage V 1 Less thanWherein v is the average moving speed of the microorganism, m is the average mass of the microorganism, and q is the charge amount of the microorganism. Thus, the contact of the microorganism with the inner wall of the first detection channel is avoided.
In some embodiments, the electrode pairs are disposed opposite each other on either side of the first detection channel sidewall; as shown in fig. 3, the distance between two electrodes of the electrode pair is d, the electrode length along the flow direction is l, the electrode equivalent area is a, and the distance from the central axis of the microfluidic channel to the wall surface of the channel is r.
In some embodiments, the voltage V 2 Greater thanWherein p is the pressure of the fluid in the first detection channel, q is the charge quantity of the microorganism, as is the sectional area of the first detection channel, and mu is the friction coefficient between the fluid and the wall surface of the microfluidic channel. If a voltage V is used 2 Is greater than->Microorganisms can be adsorbed on the electrode; microorganisms can be desorbed if the electrode voltage is reduced or a reverse voltage is provided. Therefore, the flow process of microorganisms in the microfluidic channel can be regulated and controlled, and the reliability and the precision of automatic microfluidic sorting are further improved.
In some embodiments, the determination of whether the microorganisms to be tested are agglomerated is made by: according to the formulaCalculating the number of microorganisms, wherein I: a current; t: time; Δε: a change in permittivity caused by a single cell; a: equivalent area of electrode；V 1 : voltage value. Thus, when the microorganism passes through the single electrode pair, the capacitance in the electrode pair changes, and a constant weak voltage V is used, because the permittivity of the microorganism is different from that of the bacterial liquid solvent when the microorganism is used for capacitance detection 1 Detecting the capacitance change, when the capacitance of one electrode pair changes, determining that the electrode pair has microorganisms passing through, further determining the microorganism position, and estimating the microorganism number n passing through the electrode pair.
In some embodiments, a number of microorganisms greater than 1 is an indication that the microorganisms to be tested are agglomerated.
In some embodiments, a number of microorganisms of 1 or less is an indication that no agglomeration of the microorganisms to be tested has occurred. Thus, when the number n of microorganisms is 1 or less, the microorganisms to be tested are regarded as single microorganisms, and the subsequent identification of the microorganisms can be continued.
In some embodiments, in step S100, further comprising: and acquiring image information of the microorganisms to be detected in the first detection channel, and primarily identifying the types of the microorganisms to be detected based on the image information. Therefore, the number n of the microorganisms to be detected can be further judged through the image information, so that the accuracy of detecting the number of the microorganisms to be detected is improved. Meanwhile, the morphology of the microorganism may be analyzed based on the image information to determine whether it is a target microorganism. If it is determined clearly that the microorganism is not the target microorganism, the microorganism is discarded.
The image information may be obtained using a CCD image sensor package, for example.
In some embodiments, the outlet of the first detection channel is respectively communicated with the inlet of the target microorganism channel and the inlet of the waste liquid channel, and the inner diameter of the inlet of the target microorganism channel is smaller than the inner diameter of the inlet of the waste liquid channel; the outlet of the target microorganism channel is communicated with the inlet of the second detection channel. Thus, if the number of microorganisms to be detected in the first detection channel is greater than 1, introducing the microorganisms into the waste liquid channel; if the number of the microorganisms to be detected in the first detection channel is less than or equal to 1, introducing the microorganisms into the target microorganism channel and entering the second detection channel. The inner diameter of the inlet of the target microorganism channel is smaller than that of the inlet of the waste liquid channel, so that the agglomerated microorganisms can directly enter the waste liquid channel.
In some embodiments, in step S200, further comprising: pressure is applied to the outlet of the first detection channel to allow non-agglomerated microorganisms to pass through the target microorganism channel into the second detection channel. Thus, when non-agglomerated microorganisms are at the inlet of the target microorganism channel, the non-agglomerated microorganisms can be introduced into the target microorganism channel by applying pressure.
In some embodiments, as shown in fig. 4, prior to step S100, the method further comprises:
s000, pretreatment: the bacterial liquid is diluted in advance. Therefore, impurities except microorganisms in the bacterial liquid can be further reduced, and the microorganism separation and culture efficiency is improved.
Illustratively, the bacterial liquid of the microorganism to be detected is diluted to a microorganism density of less than 10 4 And each mL.
The pretreatment method includes, but is not limited to, buffer washing, dilution, centrifugation, and sieving.
S200, raman identification: and introducing the microorganisms which are not agglomerated into a second detection channel of the Raman identification device, emitting laser to the second detection channel, recording Raman signals, drawing a spectrogram based on the Raman signals, comparing the spectrogram with the spectrogram of the known microorganisms, and determining the type of the microorganisms to be detected.
According to the embodiment of the invention, the Raman identification device can be used for carrying out real-time in-situ, high-flux and intelligent automatic identification on the microorganisms which are not agglomerated in the bacterial liquid of the microorganisms to be detected, and the type of the microorganisms is determined by analyzing the identification result and the spectrogram of the known microorganisms so as to screen out target microorganisms, and the target microorganisms can be directly subjected to targeted culture in the follow-up process, so that the method has the advantages of improving the culture efficiency, realizing specific culture, reducing the cost and the like, and can provide a new mode for microorganism research and future diagnosis and treatment.
In some embodiments, the raman authentication device comprises a plurality of second detection channels, and the laser wavelength emitted to each second detection channel is different.
The inventors have found through a number of experiments that raman scattering is inelastic scattered light generated by photons impinging on the surface of a substance, which inelastic scattered light undergoes a defined energy change compared to the incident light, resulting in a raman spectrum having a defined energy shift, which is related to the absolute wavelength and the excitation wavelength. Based on the above, the inventor uses different array laser wavelengths for different channels, and under the condition that the wave numbers are calculated by taking any excitation wavelength as a reference, the raman spectrums of microorganisms acquired by different channels are located in different wave number regions. Therefore, one spectrometer can obtain Raman spectra of microorganisms in different channels at one time, and signal decoupling is realized.
In some embodiments, a raman identification tag is disposed in each of the second detection channels. Therefore, the multi-channel signal decoupling can be further assisted, and the decoupling precision is improved.
In some embodiments, the second detection channel is serpentine. Thus, since the laser range can be irradiated to a plurality of positions in the serpentine channel, when the microorganism passes through the serpentine channel of the Raman identification area, the Raman signal can be continuously and dynamically captured even if the microorganism continuously moves, so that the Raman integration time is prolonged under the non-fixed and flowing conditions.
In some embodiments, a voltage is applied at the inlet of the second detection channel to control the flow rate and/or time of non-agglomerated microorganisms into the second detection channel. Therefore, before the start of Raman identification, the microorganisms can be controlled and released simultaneously by the multiple channels, and the multiple channel detection signals are ensured to have sufficient integration time, so that the effect of subsequent Raman spectrum detection on the microorganisms is further improved.
In some embodiments, a plurality of electrode pairs are disposed within the second detection channel. Therefore, the matching degree of the laser array movement and the microorganism movement can be improved in a correcting way, and the signal quality is further improved.
In some embodiments, as shown in fig. 4, after step S200, the method further comprises: s400, targeted culture: the determined type of microorganism to be tested is cultivated. Therefore, the target microorganism can be subjected to targeted culture by adopting proper culture conditions aiming at the characteristics of the target microorganism, so that the survival rate of the target microorganism in culture is improved.
In some embodiments, as shown in fig. 4, after step S200 and before step S300, the method further comprises: s300, automatic sorting: sorting the determined type of microorganisms to be detected, conveying target microorganisms to the step S400 for culturing, and conveying non-target microorganisms to the waste liquid recovery device for collection.
System for identifying microorganisms
In yet another aspect of the invention, the invention provides a system for performing the above method of identifying microorganisms. The system comprises: the preliminary identification device 100, the preliminary identification device includes a first detection channel 110, and an electrode pair is disposed on the first detection channel 110; a raman authentication device 200 comprising a second detection channel 210 and a laser emitting member adapted to emit laser light into the second detection channel 210; a power supply device connected to the electrode pair on the first detection channel 110; the information collecting device is suitable for collecting the electric signals of the electrode pairs; an optical signal collecting device 300 adapted to collect the laser signal of the specific position irradiated by the laser emitting part; and the analysis device 400 is respectively connected with the electric signal collection device and the optical signal collection device.
By adopting the system, bacterial liquid of microorganisms to be detected is guided into the preliminary identification device, the electric signal collection device receives electric signal information detected in the preliminary identification device and sends the electric signal information to the analysis device for analysis, the analysis device judges whether the microorganisms passing through the first detection channel are agglomerated, then the microorganisms which are not agglomerated are guided into the Raman identification device for spectral detection, the optical signal collection device receives spectral information detected in the Raman identification device and sends the spectral information to the analysis device for analysis, so that the type of the microorganisms is determined, target microorganisms can be screened later, and target cultivation can be directly carried out according to the identification result, so that the system has the advantages of improving cultivation efficiency, realizing specific cultivation, reducing cost and the like, and a new mode can be provided for microorganism research and future diagnosis.
The system for identifying microorganisms according to the present invention will be described in detail with reference to FIG. 5.
The system of the present invention comprises a preprocessing device 000, a preliminary authentication device 100, a raman authentication device 200, a power supply device, an electric signal collection device, an optical signal collection device 300, a control part 500, a culture device 600, and an analysis device 400.
In some embodiments, the preliminary authentication device 100 includes a first detection channel 110 and an image recognition component 120. At least one group of electrode pairs are arranged on the first detection channel 110, and when microorganisms pass through the first detection channel 110, voltage signal changes are obtained by acquiring capacitance changes between the electrode pairs; moreover, through the arrangement of the electrode pairs, the voltage of the electrode pairs in the first detection channel can be controlled, so that the contact between microorganisms and the inner wall of the first detection channel can be avoided, and the adsorption and desorption of the microorganisms can be realized. And based on capacitance detection and electrode adsorption, the dynamic tracking and regulation of the microorganism in the whole process with low cost can be realized.
In some embodiments, the electrode pairs are disposed opposite each other on either side of the first detection channel 110.
In some embodiments, the image recognition component 120 is disposed on the first detection channel 110, and is configured to obtain image information of the microorganism to be detected, and further determine the number n of the microorganism to be detected, so as to improve the accuracy of detecting the number n of the microorganism to be detected.
Illustratively, the image recognition component 120 is selected from a CCD image sensor.
In some embodiments, the first detection channel 110 is connected to a target microorganism channel and a waste channel, respectively, and the inlet of the target microorganism channel and the inlet of the waste channel are both connected to the outlet of the first detection channel 110, and the inner diameter of the inlet of the target microorganism channel is smaller than the inner diameter of the inlet of the waste channel; the outlet of the target microorganism channel is connected to the inlet of the first detection channel 110, and the outlet of the waste liquid channel is installed with the waste liquid recovery device 700. Thus, if the number of microorganisms to be detected in the first detection channel 110 is greater than 1, the microorganisms are introduced into the waste channel; if the number of the microorganisms to be detected in the first detection channel 110 is 1 or less, the microorganisms are introduced into the target microorganism channel and then enter the first detection channel 110. The inner diameter of the inlet of the target microorganism channel is smaller than that of the inlet of the waste liquid channel, so that the agglomerated microorganisms can directly enter the waste liquid channel.
In some embodiments, as shown in fig. 6, a pressurizing device 800 is provided at the outlet of the first detection channel 110, and when non-agglomerated microorganisms are at the inlet of the target microorganism channel, the non-agglomerated microorganisms can be introduced into the target microorganism channel by applying pressure.
Illustratively, as shown in FIG. 6, the pressurizing device 800 includes a pressure regulating device and a pressure channel.
In some embodiments, as shown in fig. 1, raman authentication device 200 includes at least one second detection channel 210 and a laser emitting component 230 that is adapted to emit laser light into second detection channel 210.
In some embodiments, a raman identification tag is further disposed in the second detection channel 210, which can further assist in multi-channel signal decoupling and improve decoupling accuracy.
In some embodiments, at least one electrode pair is disposed in the second detection channel 210, and the electrode pair is connected to a power supply device, so as to correct and improve the matching degree between the laser array movement and the microorganism movement, and further improve the signal quality.
In some embodiments, as shown in FIG. 8, the electrode pairs are disposed opposite each other on either side of the second detection channel 210. It should be noted that the electrode pairs may be located on the left and right sides of the bottom concave surface of the second detection channel 210, or may be located on the upper and lower right sides of the bottom concave surface of the second detection channel 210, and are not particularly limited.
In some embodiments, as shown in fig. 7, the second detection channel 210 has a serpentine shape with a concave bottom, and the focal position of the concave surface coincides with the focal point of the laser emitting part 230.
In some embodiments, as shown in fig. 1, the optical signal collecting device 300 is used to collect the laser signal of a specific location irradiated by the laser emitting part 230.
Illustratively, the laser emitting part 230 in the present embodiment may be a plurality of lasers.
In some embodiments, the system of the present invention may further include an optical path coupling device 220, where the optical path coupling device 220 may focus the laser generated by the laser emitting component 230 in the second detection channel 210, for detecting the raman spectrum of the microorganism to be detected located in the second detection channel 210, and the optical path coupling device 220 uses a scanning galvanometer, a half mirror, etc. to make the laser emitted by the laser emitting component 230 form a large-spot array laser.
It should be noted that, the optical path coupling device 220 may also be used as the optical signal collecting device 300, and collect the raman scattered signal generated in the raman detection area.
In some embodiments, as shown in fig. 1, the system of the present invention may further include a raman spectrum signal processor 310, where the optical signal collecting device 300 transmits the raman spectrum signal to the raman spectrum signal processor 310, and the raman spectrum signal processor 310 may further obtain the spectrum signals coupled by the plurality of second detection channels 210.
It should be noted that, the mode of the large-spot array laser form includes, but is not limited to: dividing one laser beam into a plurality of beams, and fixedly focusing the beams at a plurality of positions to form a light spot array; the laser spot is movable within a certain range to form a spot detection area.
The inventor can continuously and dynamically capture the Raman signal of the microorganism while the microorganism continuously moves through the serpentine structure of the second detection channel 210 and the arrangement of the large-spot array laser, so that the Raman integration time is prolonged under the non-fixed and flowing conditions, the microorganism is not required to be fixed during detection, the microorganism deformation is avoided, and the subsequent culture activity of the microorganism is prevented from being influenced; in addition, the invention does not need to use electron multiplication CCD (EMCCD) to detect Raman spectrum, and has the advantages of high Raman detection signal-to-noise ratio and the like.
In some embodiments, as shown in FIG. 1, the second detection channels 210 have at least two, and the wavelengths within different second detection channels 210 are also different. And under the condition that different array laser wavelengths are used for different channels and wave numbers are calculated by taking any excitation wavelength as a reference, the Raman spectra of microorganisms acquired by different channels are positioned in different wave number areas, so that one spectrometer can be used for measuring the Raman spectra of microorganisms in different channels at one time, and signal decoupling is realized.
It should be noted that the number of the second detection channels 210 in the system of the present invention may be plural, and when the number of the detection channels increases, the number of lasers needs to be increased correspondingly, and it is ensured that each of the lasers used has a different wavelength.
Illustratively, as shown in fig. 9, the second detection channels 210 are three, the lasers are three, the laser wavelengths of the two lasers are different, and the raman signal wavelengths obtained by the three second detection channels 210 are different.
In some embodiments, as shown in fig. 1, the outlet of the second detection channel 210 is connected to a target microorganism channel and a waste liquid channel, respectively, and the inlet of the target microorganism channel and the inlet of the waste liquid channel are both connected to the outlet of the second detection channel 210, and the inner diameter of the inlet of the target microorganism channel is smaller than the inner diameter of the inlet of the waste liquid channel; the outlet of the target microorganism channel is connected to the culture device 600, and the waste liquid recovery device 700 is installed at the outlet of the waste liquid channel. Thus, if the microorganism to be detected in the second detection channel 210 is a non-target microorganism, the microorganism is introduced into the waste liquid channel; if the microorganism to be detected in the second detection channel 210 is a target microorganism, the microorganism is introduced into the target microorganism channel and then enters the second detection channel 210.
In some embodiments, as shown in fig. 6, a pressurizing device 800 is provided at the outlet of the second detection channel 210, and the target microorganism can be introduced into the target microorganism channel by applying pressure when the target microorganism is at the inlet of the target microorganism channel.
Illustratively, as shown in FIG. 6, the pressurizing device 800 includes a pressure regulating device and a pressure channel.
In some embodiments, as shown in FIG. 1, the culture device 600 is coupled to the Raman identification device 200. Thus, the target microorganism can be targeted for cultivation, so that the survival rate of the target microorganism can be improved.
In some embodiments, the control part 500 is connected to the preliminary authentication device 100, the raman authentication device 200, the power supply device, the electric signal collection device, the optical signal collection device 300, the analysis device 400, the image recognition part, the pressurizing device, and the culturing device 600, respectively. Therefore, intelligent automatic control of the system can be realized.
In some embodiments, the preprocessing unit 000 is coupled to the inlet of the first detection channel 110. Thus, the pretreated bacterial liquid can directly enter the first detection channel 110 for separation.
In the system disclosed in one embodiment of the present invention, as shown in fig. 5, after the bacterial liquid of the microorganism to be detected is diluted by the preprocessing device 000, the bacterial liquid is introduced into the first detection channel 110, the capacitance signal and the microorganism image obtained by the preliminary identification device 100, and the spectrum signal obtained by the raman spectrum signal processor 310 and coupled by the plurality of channels, and the capacitance signal, the microorganism image and the spectrum signal are all input into the analysis device 400 for data analysis, and because the wavelengths of the laser light generated by different lasers are different, the wavelengths of the raman signals obtained by different second detection channels 210 are different, and meanwhile, raman signal markers are respectively arranged in the different second detection channels 210, and the data analysis device 400 can implement the decoupling of the raman spectrum signals of different second detection channels 210 based on the signal markers and the laser light wavelengths of the lasers.
Based on the capacitance information, the image information, and the raman spectrum information, the data analysis device 400 judges whether the detected microorganism is a target microorganism, and if not, the control part 500 drives the preliminary authentication device 100 or the raman authentication device 200 so that the microorganism enters the waste liquid recovery device 700; in contrast, if the microorganism is the target microorganism, the control unit 500 drives the preliminary identification device 100 to allow the microorganism to enter the raman identification device 200, and finally allows the microorganism to enter the intelligent targeted culture device 600.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: method for identifying microorganisms
The detection steps are as follows:
s000, pretreatment: the bacterial liquid is diluted in advance.
S100, preliminary sorting: and (3) introducing bacterial liquid containing microorganisms to be detected into a first detection channel of the preliminary identification device, applying voltage to the side wall of the first detection channel, recording a voltage signal, judging whether the microorganisms to be detected are agglomerated or not based on the change of the voltage signal, and collecting the microorganisms which are not agglomerated.
S200, raman identification: and introducing the microorganisms which are not agglomerated into a second detection channel of the Raman identification device, emitting laser to the second detection channel, recording Raman signals, drawing a spectrogram based on the Raman signals, comparing the spectrogram with the spectrogram of the known microorganisms, and determining the type of the microorganisms to be detected.
The results are shown in FIG. 10, and analyzed with reference to the Raman spectra of the microorganisms shown in FIG. 11, the microorganisms in the three channels in FIG. 10 were Lactobacillus paracasei (channel 1), bacillus subtilis (channel 2) and Bacillus subtilis (channel 3), respectively.
The method and system of the present invention have the following advantages over the prior art:
1. the inventor firstly puts forward an identification-screening-culture technical route, and can realize non-contact, rapid and real-time identification of microorganism bacteria through capacitance signals and map signals, and realize high-throughput rapid detection and single microorganism separation; by adopting a large-spot array laser measurement mode to continuously track and detect single microorganisms in a channel, a high-quality Raman spectrum signal can be obtained under a dynamic detection condition, the defect of the prior single-cell Raman identification technical means is overcome, and the method is simple and easy to popularize; by adopting a multi-wavelength measurement mode, the decoupling detection of the multi-channel sample can be realized, the measurement speed is remarkably improved, and the rapid, low-cost and high-flux identification is realized; through the sorting technology, the identified microorganisms are automatically screened and the specific culture is carried out based on big data analysis, so that the automation and the intellectualization of the microorganism separation culture are realized, the preculture is not needed, the strain separation efficiency can be effectively improved, and the average culture cost is reduced.
2. The invention uses the electrode pair as a sensor and a controller, only the voltage at two ends of the electrode pair is required to be changed, namely, the switch between the sensor and the controller can be realized, namely, the microorganisms are detected through capacitance change, and the flow speed and/or time of the microorganisms can be controlled through the adsorption and the desorption of the microorganisms, so that the dynamic tracking and regulation of signals of the whole process can be realized under the condition of low cost, the regulation and control capability of a microfluidic system on the microorganisms is obviously improved through the judgment of the number and the flow speed of the microorganisms and the controllable capture and release, the feasibility of the multi-wavelength light spot dynamic Raman detection of single microorganisms in a channel is ensured, and the sorting success rate is effectively improved.
3. The invention synthesizes the detection results of the Raman spectrum signal, the image signal and the capacitance signal to obtain the target microorganism, can realize the rapid identification of the microorganism in the mixed microorganism solution such as the fecal bacterial suspension and the like under the condition of no need of pre-culture, can be used for identifying the infectious disease source, can greatly shorten the time required by diagnosis, and has important significance for medical diagnosis and treatment.
4. The system provided by the invention has more than two lasers with different wavelengths, can perform dual-wavelength measurement on the same microorganism, realizes the decoupling of fluorescence and Raman signals, solves the problem of strong fluorescence background interference of biological samples in principle, avoids uncertainty caused by manually eliminating fluorescence, can eliminate instrument system errors, remarkably improves the precision, and can provide a reliable identification reference for a microorganism-Raman spectrum database; and by processing Raman detection signals of Raman identification markers in different second detection channels, the influence of laser power and wavelength of different channels can be further reflected, and based on Raman signal references of the identification markers, under the condition that the multichannel detection signals have intersections, the multichannel signal decoupling precision is further improved by performing rough fitting on measurement spectrums of non-intersection positions and database spectrums, combining with correction of Raman signal intensity ratio of the markers, and iteratively decoupling multichannel Raman spectrum signals. Therefore, the non-contact type single microorganism rapid identification and character analysis can be realized, and the automation of the microorganism culture process is greatly promoted.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method of identifying a microorganism, comprising:
(1) Introducing bacterial liquid containing microorganisms to be detected into a first detection channel of a preliminary identification device, applying voltage to the side wall of the first detection channel, recording a voltage signal, judging whether the microorganisms to be detected are agglomerated or not based on the change of the voltage signal, and collecting the microorganisms which are not agglomerated;
wherein the applying of the voltage is performed by a pair of electrodes on the side wall of the first detection channel;
judging whether the microorganisms to be tested are agglomerated or not by the following method:
according to the formulaCalculating the number of microorganisms, wherein d: the distance between the electrode pair and the two electrodes; i: a current; t: time; Δε: a change in permittivity caused by a single cell; a: electrode equivalent area; v (V) 1 : a voltage value;
the microorganism number being greater than 1 is an indication of agglomeration of the microorganism to be tested;
the microorganism number being less than or equal to 1 is an indication that agglomeration of the microorganisms to be detected has not occurred;
(2) And introducing the microorganisms which are not agglomerated into a second detection channel of a Raman identification device, emitting laser to the second detection channel, recording a Raman signal, drawing a spectrogram based on the Raman signal, comparing the spectrogram with a spectrogram of a known microorganism, and determining the type of the microorganism to be detected.
2. The method according to claim 1, wherein the bacterial liquid is subjected to dilution treatment in advance before the step (1);
after step (2) is carried out, the determined type of microorganism to be tested is cultivated.
3. The method of claim 1, wherein in step (1), further comprising: and acquiring image information of the microorganisms to be detected in the first detection channel, and primarily identifying the types of the microorganisms to be detected based on the image information.
4. The method of claim 1, wherein the outlet of the first detection channel is in communication with an inlet of a target microorganism channel and an inlet of a waste channel, respectively, the inlet inner diameter of the target microorganism channel being smaller than the inner diameter of the waste channel inlet; the outlet of the target microorganism channel is communicated with the inlet of the second detection channel;
the method further comprises: applying pressure to the outlet of the first detection channel to cause the non-agglomerated microorganisms to pass through the target microorganism channel into the second detection channel.
5. The method of claim 1, wherein the raman authentication device comprises a plurality of second detection channels, and wherein the laser wavelength emitted to each of the second detection channels is different, and wherein the second detection channels have a serpentine shape.
6. The method of claim 1, wherein a voltage is applied at the inlet of the second detection channel to control the flow rate and/or time of the non-agglomerated microorganisms into the second detection channel.
7. A system for performing the method for identifying microorganisms according to any one of claims 1 to 6, comprising:
the primary identification device comprises a first detection channel, and an electrode pair is arranged on the first detection channel;
a raman authentication device comprising a second detection channel and a laser emitting component adapted to emit laser light into the second detection channel;
the power supply device is connected with the electrode pair on the first detection channel;
an electrical signal collection device adapted to collect electrical signals of the electrode pairs;
an optical signal collection device adapted to collect a laser signal of a specific position irradiated by the laser emitting part;
and the analysis device is respectively connected with the electric signal collection device and the optical signal collection device.
8. The system of claim 7, wherein the first detection channel is further provided with an image recognition component;
the outlet of the first detection channel is respectively communicated with the inlet of the target microorganism channel and the inlet of the waste liquid channel, and the inner diameter of the inlet of the target microorganism channel is smaller than that of the inlet of the waste liquid channel; the outlet of the target microorganism channel is communicated with the inlet of the second detection channel;
the outlet of the first detection channel is provided with a pressurizing device;
an electrode pair is arranged on the inlet of the second detection channel, and the electrode pair is connected with the power supply device;
the system further comprises: and the culture device is connected with the Raman identification device.
9. The system of claim 8, wherein the raman authentication device comprises a plurality of the second detection channels, wherein the second detection channels are serpentine in shape and have a concave bottom, and a focal position of the concave bottom coincides with a focal point of the laser emitting member;
the system further comprises:
and the control part is respectively connected with the preliminary identification device, the Raman identification device, the power supply device, the electric signal collection device, the optical signal collection device, the analysis device, the image identification part, the pressurizing device and the culture device.
10. The system of claim 8, wherein the image recognition component is a CCD image sensor.
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