An automated rapid detection instrument and method for microbial Raman drug resistance

Abstract

The present invention relates to an automated rapid detection instrument and detection method for microbial Raman drug resistance. The device includes an electric displacement platform, an imaging module, an excitation light module, a micro-focusing module, a Raman main optical path and transmission module, a coaxial illumination module, and an automatic acquisition and control module; the electric displacement platform is used to place the sample chip; the excitation light module is used to emit laser light; the micro-focusing module is used to automatically focus the laser light on the to-be-detected dental plaque at the optimal acquisition position, so that the to-be-detected dental plaque generates Raman signals; the coaxial illumination module is used to provide coaxial illumination light for the micro-focusing module; the Raman main optical path and transmission module is used to obtain the Raman spectrum of the to-be-detected dental plaque; the automatic acquisition and control module is used to control the excitation light module, the Raman main optical path and transmission module, the micro-focusing module, the coaxial illumination module, the imaging module and the electric displacement platform, and process the Raman spectrum of the to-be-detected dental plaque, so as to realize the automated detection of the to-be-detected dental plaque and the rapid identification of drug resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to an automated microbial Raman drug resistance rapid detection instrument and a detection method. Background Art

[0002] Clinically, the traditional detection of pathogenic bacteria drug resistance mainly uses the culture method, which has the disadvantages of long detection time, high professional requirements for experimental operators, and easy occurrence of false positives. To contain the spread of drug resistance, it is necessary not only to develop new antibiotics, but also to develop rapid drug resistance detection instruments and methods to improve the pertinence and effectiveness of the use of existing antibiotics, thereby delaying and containing the spread of drug resistance.

[0003] Currently, one of the most promising directions is the "single-cell" drug resistance detection technology, that is, skipping cell culture proliferation and directly characterizing the "growth" or "metabolism" phenotypes of the original single cells in the sample with single-cell precision, achieving the goals of rapidity, phenotype-based, and wide applicability in principle.

[0004] Raman spectroscopy is an efficient information recognition technology. By analyzing the inelastic scattering spectrum lines of compounds by specific incident light, the vibrational or rotational energy levels of compound molecules can be directly detected. By analyzing the Raman characteristic spectrum lines, information on the molecular composition and structure of compounds can be obtained. Existing detection instruments using Raman technology can only detect single-cell samples, are applicable to slow-growing and difficult-to-culture microorganisms, cannot quickly locate the sample position, have slow detection speed and low efficiency, and cannot detect dental plaque. Summary of the Invention

[0005] Aiming at the above problems, the purpose of the present invention is to provide an automated microbial Raman drug resistance rapid detection instrument and a detection method, which can detect the dental plaque of microbial cells, quickly locate the position of the dental plaque, and realize the automated rapid detection of microbial samples.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] An automated microbial Raman drug resistance rapid detection instrument includes an electric displacement platform, an imaging module, an excitation light module, a microscopic focusing module, a Raman main optical path and transmission module, a coaxial illumination module, and an automatic acquisition and control module;

[0008] The electric displacement platform is used to place the sample chip;

[0009] The imaging module is used to photograph the panoramic information of the sample chip and the microscopic information of the dental plaque, and cooperate with the electric translation platform to quickly locate the to-be-detected dental plaque on the sample chip and determine the acquisition position of the to-be-detected dental plaque;

[0010] The excitation light module is used to emit laser light;

[0011] The microscopic focusing module is used to automatically focus the laser light on the dental plaque to be measured at the acquisition position, so that the dental plaque to be measured generates Raman signals;

[0012] The coaxial illumination module is used to provide coaxial illumination light for the microscopic focusing module;

[0013] The Raman main optical path and transmission module is used to obtain the Raman spectrum of the dental plaque to be measured;

[0014] The automatic acquisition control module is used to control the excitation light module, the Raman main optical path and transmission module, the microscopic focusing module, the coaxial illumination module, the imaging module and the electric displacement platform, and process the Raman spectrum of the dental plaque to be measured, so as to realize the automatic detection of the dental plaque to be measured.

[0015] Preferably, the automatic microbial Raman drug resistance rapid detection instrument includes an image observation mode and a Raman measurement mode;

[0016] The image acquisition mode is used to obtain the image information of the sample chip;

[0017] The Raman measurement mode is used to realize the acquisition of the Raman information of the dental plaque to be measured on the sample chip.

[0018] Preferably, the excitation light module includes a laser, an electric shutter, a beam expander and an electrically tunable attenuator; the Raman main optical path and transmission module includes a broadband mirror, a dichroic mirror, a second lens, a pinhole, a microscopic objective lens, a spectrometer and a detector;

[0019] The laser light emitted by the laser sequentially passes through the electric shutter, the beam expander, the electrically tunable attenuator, the mirror, the dichroic mirror and the broadband mirror and is reflected to the microscopic objective lens to be focused on the sample chip on the electric displacement platform;

[0020] The Raman signal light generated by the excitation of the sample chip sequentially passes through the microscopic objective lens, the broadband mirror, the dichroic mirror, the second lens, the pinhole and the spectrometer and is emitted to the detector.

[0021] Preferably, the bandwidth range of the broadband mirror is 100 nm to 200 nm plus the excitation wavelength of the laser.

[0022] Preferably, the coaxial illumination module includes an LED light source and a semi-transmissive semi-reflective mirror, and the imaging module includes a CCD camera and a first lens;

[0023] The white light emitted by the LED light source sequentially passes through the semi-transparent and semi-reflective mirror and the microscopic objective lens and is focused on the sample chip; the reflected light formed by the LED light source irradiating on the sample chip enters the CCD camera through the microscopic objective lens, the semi-transparent and semi-reflective mirror, and the first lens to form an image of the sample chip.

[0024] Preferably, the excitation light module, the Raman main optical path and transmission module, the coaxial illumination module, and the imaging module are fixed inside the same optical box body. The optical box body is a fully enclosed structure, and the Raman main optical path and transmission module and the microscopic focusing module are located in the same plane.

[0025] Preferably, the detection instrument further includes a sterilization module for eliminating microorganisms in the environment to prevent cross-contamination.

[0026] A detection method for an automated microbial Raman drug resistance rapid detection instrument includes the following steps:

[0027] The imaging module captures an image of the entire sample chip to obtain the position information of each to-be-detected plaque.

[0028] Move the electric displacement platform to the first to-be-detected plaque to determine the best collection position of the to-be-detected plaque.

[0029] The excitation module emits a laser, and the laser is automatically focused on the to-be-detected plaque through the microscopic focusing module, so that the to-be-detected plaque generates a Raman signal.

[0030] The Raman main optical path and transmission module acquire the Raman spectrum of the plaque and complete the detection of the plaque through the automatic acquisition control module.

[0031] Move to the next to-be-detected plaque of the electric displacement platform and repeat the above process, process the Raman spectrum data of each to-be-detected plaque, and complete the drug resistance detection.

[0032] Preferably, determining the best collection position of the to-be-detected plaque includes:

[0033] Collect the plaque image.

[0034] Convert the image processing into a grayscale image and perform filtering processing.

[0035] Judge the image pixel points. If it is higher than the set threshold, the image pixel point is a bright spot; if it is lower than the set threshold, check whether there is a plaque at its surrounding position.

[0036] Locate the dense area of the bright spots as the position of the plaque.

[0037] Preferably, the automatic focusing process includes:

[0038] Use the hill climbing search algorithm to select multiple position points of the first to-be-detected plaque.

[0039] Use the clarity evaluation function values of the multiple position points to fit the local curves where the multiple position points are located;

[0040] Determine whether there is a peak greater than the set threshold within the curve. If there is such a peak, return the point immediately before the point closest to the maximum value among this section of position points. Starting from this point and ending with the point immediately after the maximum value point, re-divide the search range and reduce the search step size; repeat the search until the required focusing accuracy is achieved. If there is no such peak, continue to move in the original direction and repeat the above focusing operation to finally confirm the optimal focal plane.

[0041] Due to the adoption of the above technical solutions, the present invention has the following advantages: 1. The automated microbial Raman drug resistance rapid detection instrument and detection method provided by the present invention can detect the microbial cell plaque through the mutual cooperation of the microscopic focusing module, Raman main optical path and transmission module, coaxial illumination module, imaging module, electric displacement platform and automatic acquisition and control module, reducing the focusing difficulty and the requirements for the detector, being able to quickly locate the plaque position, while reducing the detection time and cost, and realizing the rapid detection of automated microbial samples, with high sensitivity and high stability. 2. The automated microbial Raman drug resistance rapid detection instrument and detection method provided by the present invention adopt Raman technology to automatically detect clinical pathogenic microbial plaque, with an integrated and fully enclosed structure, greatly improving the detection speed, liberating manpower, and being able to quickly achieve the detection of each drug resistance of each sample of pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a schematic optical path diagram of the detection instrument provided in Embodiment 1 of the present invention;

[0043] Figure 2 It is a schematic structural diagram of the optical box of the detection instrument provided in an embodiment of the present invention;

[0044] Figure 3 It is a schematic internal structure diagram of the detection instrument provided in an embodiment of the present invention;

[0045] Figure 4 It is a working flow chart of the detection method of Embodiment 2 of the present invention;

[0046] Figure 5 It is a schematic diagram of the autofocus scheme provided in an embodiment of the present invention;

[0047] Figure 6 It is a Raman spectrogram of a deuterium-labeled microbial plate provided in an embodiment of the present invention.

[0048] Each reference numeral in the figure:

[0049] 1 is a laser, 2 is an electric shutter, 3 is a beam expander, 4 is an electrically adjustable attenuator, 5 is a mirror, 6 is a dichroic mirror, 7 is a broadband mirror with a one-dimensional electric displacement stage, 8 is a microscope objective, 9 is an electric displacement platform, 10 is an LED light source, 11 is a semi-transparent and semi-reflective mirror, 12 is a first lens, 13 is a area array CCD, 14 is a second lens, 15 is a pinhole, 16 is a spectrometer, 17 is a detector, 18 is an imaging CCD; 19 is an ultraviolet lamp, 20 is an optical box; 21 is a support frame; 22 is a sample chip. Detailed implementation mode

[0050] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0051] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "front", "rear", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the system or component referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0052] In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "assembly", "installation", "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0053] The automated microbial Raman drug resistance rapid detection instrument and detection method provided by the present invention can automatically detect clinical pathogenic microbial plaques by using Raman technology, quickly realize the drug resistance detection of pathogenic bacteria, improve the detection speed, liberate human resources, and improve the detection accuracy and stability.

[0054] Next, the automated microbial Raman drug resistance rapid detection instrument and detection method provided by the embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0055] Embodiment 1

[0056] As Figure 1As shown in the figure, the automated rapid detection instrument for microbial Raman drug resistance provided in this embodiment includes an electric displacement platform, an imaging module, an excitation light module, a microscopic focusing module, a Raman main optical path and transmission module, a coaxial illumination module, and an automatic acquisition and control module;

[0057] The electric displacement platform is used to place the sample chip, and cooperate with the imaging module to achieve automatic focusing of the sample and quickly locate the position of the dental plaque;

[0058] The imaging module is used to capture the panoramic information of the sample chip, obtain the position information of each dental plaque, and form position correction information obtained by comparing each dental plaque with a preset dental plaque; at the same time, it is used to capture the microscopic image of the dental plaque, and cooperate with the electric translation platform to quickly locate the dental plaque to be tested on the sample chip and determine the collection position of the dental plaque to be tested;

[0059] The excitation light module is used to emit laser light;

[0060] The microscopic focusing module is used to automatically focus the laser on the dental plaque to be tested at the collection position, so that the dental plaque to be tested generates Raman signals. At the same time, this focusing module is also the focusing component for white light imaging;

[0061] The Raman main optical path and transmission module are used to obtain the Raman spectrum of the dental plaque and transmit the Raman spectrum information of the dental plaque to the automatic acquisition and control module;

[0062] The coaxial illumination module is used to provide coaxial illumination light for the microscopic focusing module; the Raman main optical path and transmission module are used to obtain the Raman spectrum of the dental plaque to be tested;

[0063] The automatic acquisition and control module is used to control the excitation light module, the Raman main optical path and transmission module, the microscopic focusing module, the coaxial illumination module, the imaging module and the electric displacement platform, and process the Raman spectrum of the dental plaque to be tested to achieve automatic detection of the dental plaque to be tested.

[0064] Among them, the detection instrument further includes a sterilization module, which is used to eliminate microorganisms in the environment and prevent cross-contamination;

[0065] The electric displacement platform is a three-dimensional moving platform, which can realize automatic movement in the X, Y, and Z directions.

[0066] The excitation light module includes a laser 1, an electric shutter 2, a beam expander 3, and an electrically adjustable attenuator 4.

[0067] The beam expansion ratio of the beam expander 3 is 3:1 or 4:1 or 5:1.

[0068] The electrically adjustable attenuator 4 has at least two different attenuation rates, and the electrically adjustable attenuator 4 can adjust the attenuation effect through software.

[0069] The automated microbial Raman drug resistance rapid detection instrument provided in this embodiment can include two working modes, namely, the image observation mode and the Raman measurement mode. The image acquisition mode can obtain the image information of the sample, and the Raman measurement mode mainly realizes the acquisition of the Raman information of the sample. The two working modes can be switched by a broadband mirror 7 with a one-dimensional electric displacement stage in a common optical path, so as to collect all Raman signals, achieve the optimal Raman signal collection, and the two can be quickly switched, so as to ensure the fastest acquisition of the Raman signal of the sample and reduce the time for bacterial drug resistance identification.

[0070] The broadband mirror 7 is installed on the one-dimensional electric displacement stage and placed at the optical path intersection of the main Raman optical path and transmission module and the imaging module.

[0071] The microscopic focusing module includes a microscopic objective lens 8, and the microscopic objective lens 8 is a quadruple, ten-fold, fifty-fold or one-hundred-fold compound / half-compound apochromatic objective lens.

[0072] The main Raman optical path and transmission module includes a broadband mirror 7, a dichroic mirror 6, a second lens 14, a pinhole 15, a spectrometer 16 and a detector 17.

[0073] The bandwidth range of the mirror 5 is 100 nm to 200 nm plus the excitation wavelength of the laser 1.

[0074] The dichroic mirror 6 is a high-pass and low-reflection mirror, and the reflectivity and transmittance of the dichroic mirror 6 are both higher than 90%.

[0075] The pinhole 15 is used to block the signal of stray light in the confocal case.

[0076] The spectrometer 16 is used to obtain the spectral separation of Raman scattered light.

[0077] The detector 17 is a linear array or area array CCD and is used to obtain the signal of Raman scattered light.

[0078] The coaxial illumination module includes an LED light source 10 and a semi-transmissive and semi-reflective mirror 11.

[0079] The sterilization module includes an ultraviolet lamp 19, and the ultraviolet lamp 19 is placed around the microscopic objective lens.

[0080] The imaging module can include two parts. One is an observation camera, and the observation camera can be an imaging CCD 18, and the imaging CCD 18 can realize the wide-field imaging of the entire sample chip; the other is a microscopic imaging composed of an area array CCD 13, a second lens 12 and a microscopic objective lens 8, which is used to obtain the microscopic image of the dental plaque.

[0081] Such as Figure 1As shown in the figure, for the automated rapid microbial Raman drug resistance detection instrument provided in this embodiment, a second lens 14, a pinhole 15, a spectrometer 16, and a detector 17 are sequentially arranged in the transmission light direction of the dichroic mirror 6; a semi-transmissive and semi-reflective mirror 11 is arranged in the transmission light direction of the bandwidth mirror 7, a first lens 12 and a frame CCD 13 are sequentially arranged in the transmission light direction of the semi-transmissive and semi-reflective mirror 11, and an LED light source 10 is arranged in the reflection light direction of the semi-transmissive and semi-reflective mirror 11.

[0082] The laser emitted by the laser 1 passes through the electric shutter 2, the beam expander 3, and the electrically adjustable attenuator 4 in sequence, and then is reflected by the mirror 5, the dichroic mirror 6, and the bandwidth mirror 7 with a one-dimensional electric displacement stage and enters the microscope objective 8, and finally is focused on the sample chip 22 placed on the electric displacement platform 9 to generate the Raman signal of bacteria; the Raman signal generated by the bacteria passes through the microscope objective 8, the bandwidth mirror 7 with a one-dimensional electric displacement stage, the dichroic mirror 6, the second lens 14, the pinhole 15, and the spectrometer 16 in sequence and is emitted to the detector 17, and finally forms Raman spectral data; the white light emitted by the LED light source 10 passes through the semi-transmissive and semi-reflective mirror 11 and the microscope objective 8 in sequence and is finally focused on the sample chip 22; the reflected light generated by the white light irradiating the sample passes through the microscope objective 8, the semi-transmissive and semi-reflective mirror 11, and the first lens 12 in sequence, and finally enters the frame CCD 13 camera to form an image of the sample.

[0083] Among them, the laser 1 can be one of a 532nm laser, a 633nm laser, a 785nm or a 1064nm laser, which causes little damage to cells during detection. The electric shutter 2 can quickly turn on and off the laser in the optical path, thereby avoiding the influence of the on and off of the laser due to the power off of the laser or the method of adjusting the current on the stability of the laser output. The beam expander 3 can expand the laser beam to achieve a better focusing effect. The electrically adjustable attenuator 4 can attenuate the laser intensity to different degrees, so as to quickly adjust the laser energy through software and obtain a better signal. The spectrometer 16 splits the Raman signal of the sample, and then enters the detector 17 from low to high according to the wavelength. The detector 17 can be a line array CCD, a frame CCD or an EMCCD detector, which is used to record the Raman spectral information after the spectrometer splits the light.

[0084] When measuring the Raman signal, the laser 1 emits laser light. After the intensity is adjusted by the electrically tunable attenuator 4, the laser light is projected onto the dichroic mirror 6 by the mirror 5. After being reflected by the dichroic mirror 6, the laser light is reflected by the broadband mirror 7 with a one-dimensional electric displacement stage to the objective lens and focused onto the sample. The electric displacement platform 9 can be equipped with an LED white light source to focus and image the cell sample plaque, automatically select the optimal acquisition area, and accurately focus and then collect the Raman spectrum signal. The Raman backscattering signal scattered by the cell plaque is collected by the microscope objective lens and returned to the dichroic mirror 6 through the mirror. At this time, the Raman signal can pass through the dichroic mirror 6, while other unwanted stray light including Rayleigh signal is blocked. The Raman signal is split by the pinhole 15 and the spectrometer 16 and then collected by the detector 17, and finally a Raman spectrum is formed.

[0085] As Figure 2 and Figure 3 shown, the excitation light module, the Raman main optical path and transmission module, the coaxial illumination module, and the imaging module are fixed inside the same optical box 20.

[0086] The optical box 20 includes a support frame 21, and the support frame can include three groups on the left, right, and rear, so that the optical box 20 has a preset height.

[0087] The micro-focusing module is located at the lower part of the optical box 20; the Raman main optical path and transmission module are located on one side of the optical box 20, such as the right side, and the Raman main optical path and transmission module and the micro-focusing module are located on the same plane, making the optical path more stable and the adjustment more convenient.

[0088] The optical box 20 is integrally cast and is a fully enclosed structure.

[0089] In this embodiment, the sample chip 22 is used to place the sample, and it has relatively high flatness requirements. It can be made of quartz glass slide, calcium fluoride glass slide or metal, etc. It can carry the sample and has no interfering Raman peaks in the range of 2200 - 3000 cm -1 region.

[0090] In this embodiment, the automatic acquisition control module includes a data analysis and processing device, and the data analysis and processing device can be a computer. The computer is control-connected to the laser 1, the electric shutter 2, the electrically tunable attenuator 4, the broadband mirror 7 with a one-dimensional electric displacement stage, the electric displacement platform 9, the LED light source 10, the area array CCD 13, the pinhole 15, the spectrometer 16, the detector 17, and the imaging CCD 18.

[0091] In this embodiment, the automated microbial Raman drug resistance rapid detection instrument is mainly used for the rapid detection of drug resistance of pathogenic microorganisms such as urine, gastric juice, blood, and cerebrospinal fluid.

[0092] The automatic acquisition control module mainly controls key devices such as laser 1, spectrometer 16, detector 17, and CCD 18 to achieve automatic determination of sample points, correction of sample positions, autofocus, and automatic spectrum acquisition. Autofocus needs to cooperate with the imaging module of the system and the electric displacement platform 9. The Z-axis of the electric displacement platform 9 simultaneously takes images of the sample. By using the Tenengrad gradient function as the focus evaluation function method, the evaluation function method not only retains the advantages of simple calculation and fast processing speed of the traditional gray function but also has advantages such as good unimodality and sharp and prominent peak shapes. Based on the evaluation function method, an optimal focal plane is automatically searched by developing a search algorithm. Specifically, the idea of the curve fitting method is embedded in the local search of the traditional hill climbing search algorithm to improve the focus search strategy. Specifically, during the hill climbing search process, the clarity evaluation function values of multiple position points are selected as a local curve for "fitting", and it is judged whether there is a peak greater than the set threshold within the curve. If so, it means that the best focus position (unimodal peak) must be included within the range of this section of position points. At this time, the point closest to the best focus position point (maximum value point) before this section of position points is returned. Starting from this point and ending with the point after the best focus position point, this range is re-divided and the step size is reduced to repeat the search until the required focus accuracy is achieved. If there is no peak greater than the set threshold within the curve, continue to move in the original direction and repeat the above focus operation to finally confirm the optimal focal plane.

[0093] After obtaining the optimal focal plane, due to the random appearance of plaque positions in the microscopic state, in order to quickly locate the sample points, intelligent plaque discrimination technology is developed to intelligently determine and locate the plaque and background areas. The collected images are first converted into grayscale images through image processing and then subjected to Gaussian filtering to reduce high-frequency noise. Then, the image pixel points are judged. If they are higher than the set threshold, it is considered that there is a bright spot at that place. During final positioning, the bright spot dense area is preferentially located.

[0094] After locating the plaque position, during the automated spectrum acquisition process, through real-time spectrum data analysis, as Figure 5 shown, the change diagram of the focal plane from defocus, near focus, in focus, near focus, and far focus. By intelligently adjusting acquisition parameters such as laser power, exposure time, and acquisition position, the optimal spectrum data can be obtained.

[0095] Example 2

[0096] As Figure 4 shown, the detection method of the automated microbial Raman drug resistance rapid detection instrument provided in Example 1 includes the following steps:

[0097] S01, the imaging module takes an image of the entire sample chip to obtain the position information of each plaque to be measured;

[0098] Specifically, the sample is prepared as follows:

[0099] The monoclonal sample obtained is mixed with the culture medium to establish a control group, and different concentrations and types of antibiotics and water are added respectively; in addition, an experimental group is established, and different concentrations and types of antibiotics and heavy water D2O with the same volume as the water in the control group are added to the culture medium. The control group and the experimental group are incubated for the same time. The bacterial solutions of the control group and the experimental group are pretreated and a detection chip is made. The detection chip is placed on the electric displacement platform, and the panoramic image of the sample chip is started to be taken. The panoramic image of the dental plaque is taken using the imaging CCD18 to obtain the position information of the dental plaque to be detected on the chip, and the position information of the dental plaque is determined through imaging to form the position correction information of each dental plaque and the designed dental plaque;

[0100] S02, move the electric displacement platform to the first dental plaque to be detected, and determine the best collection position of the dental plaque to be detected;

[0101] Specifically, the position of the dental plaque is identified through the corrected position information of the dental plaque, and the automatic acquisition control module controls the electric displacement platform to automatically move to the coordinate point of the first dental plaque to be detected to determine the best collection position of the dental plaque;

[0102] Specifically, a 50x objective lens is used to collect the microscopic image of the dental plaque; the image is processed and converted into a grayscale image and filtered; the image pixel points are judged. If it is higher than the set threshold, it is considered that there is a bright spot at this place; if it is lower than the set threshold, check whether there is a dental plaque at its surrounding position; the dense area of the bright spots is located as the position of the dental plaque;

[0103] Specifically, the hill climbing search algorithm is used to select multiple position points of the first dental plaque to be detected; the sharpness evaluation function value is used to fit the local curve where the multiple position points are located; judge whether there is a peak greater than the set threshold in the curve. If there is such a peak, return the point before the point closest to the maximum value among this section of position points. Taking this point as the starting point and the point after the maximum value point as the end point, re-divide the search range and reduce the search step size; repeat the search until the required focusing accuracy is achieved. If there is no such peak, continue to move in the original direction and repeat the above focusing operation to finally confirm the optimal focal plane;

[0104] S03, the excitation module emits a laser, and the laser is automatically focused on the dental plaque to be detected through the microscopic focusing module, so that the dental plaque to be detected generates a Raman signal;

[0105] Specifically, at the position of the dental plaque determined by locating the dense area of the bright spots as the position of the dental plaque, the sample is automatically focused;

[0106] S04, the Raman main optical path and transmission module acquire the Raman spectrum of the dental plaque, and complete the detection of the dental plaque through the automatic acquisition control module;

[0107] Specifically, the Raman main optical path and the transmission module are used to collect the Raman spectrum of the dental plaque, and the spectral data is analyzed in real time to determine whether the spectral data that meets the analysis requirements is obtained; if so, the acquisition parameters are automatically and intelligently adjusted to obtain the optimal spectral data; the acquisition parameters include one or more of the laser power, exposure time, and acquisition position; the number of collected Raman spectra is 5-40; if not, according to the panoramic view of the chip taken by the imaging CCD18 in step, the position information of each dental plaque to be measured is obtained, and the position information determined in the position correction information of each dental plaque and the designed dental plaque is formed, and it is automatically moved to the next dental plaque, and steps are repeated to determine the best acquisition position of the dental plaque until step to automatically collect the Raman spectrum;

[0108] S05, move to the next dental plaque on the electric displacement platform and repeat the above process to process the Raman spectral data of each dental plaque to be measured and complete the drug resistance detection.

[0109] Specifically, the automatic acquisition control module controls the next dental plaque on the electric displacement platform, repeats the above process, completes the acquisition and processing of the Raman spectral data of each dental plaque to be measured. The automatic acquisition control module determines whether the sample is drug-resistant by automatically calculating the ratio of C-D / (C-D + C-H) for the optimal spectral data of each dental plaque to be measured after the spectral data is obtained, and performs automatic data processing and automatically generates a detection report. As Figure 6 shown, calculate the area under the Raman spectrum centered at 2180 cm -1 and with a width of at least 85 cm -1 moved to the left and right from 2180 cm -1 as the carbon-deuterium peak area (C-D), and calculate the area under the Raman spectrum centered at 2940 cm -1 and with a width of at least 85 cm -1 moved to the left and right from 2940 cm -1 as the carbon-hydrogen peak area (C-H). The drug resistance index of clinical microorganisms is reflected by the degree of deuteration: D% = (C-D) / (C-D + C-H).

[0110] The detection method in the above embodiments can be used for the rapid detection of drug resistance of clinical Mycobacterium tuberculosis in sputum.

[0111] Specifically, the drug resistance of Mycobacterium tuberculosis is measured by Raman under a 50-fold objective lens, and the detection parameters are: exposure time 1 s, laser wavelength 532 nm, energy 80 mW, spectral range 500-3500 cm -1 . Raman spectra of about 30 different positions of bacterial cell dental plaques are randomly measured and collected in different fields of view for each sample.

[0112] During data processing, the collected Raman spectra are processed by background removal, baseline normalization, and maximum value standardization. Analyze the C-D peak area (in the range of 2050-2300 cm in the Raman spectrum) and the C-H area (in the range of 2800-3050 cm in the Raman spectrum), calculate the C-D ratio at different drug concentrations, and subtract the C-D ratio of the group without isoniazid / rifampicin added from the C-D ratio of the group with isoniazid / rifampicin added after incubating for 24 h, so as to calculate the ΔC-D ratio. -1 region) and the C-H area (in the range of 2800-3050 cm in the Raman spectrum) -1 ), calculate the C-D ratio at different drug concentrations, subtract the C-D ratio of the group without isoniazid / rifampicin added from the C-D ratio of the group with isoniazid / rifampicin added after incubating for 24 h, so as to calculate the ΔC-D ratio.

[0113] The results show that the effects of rifampicin and isoniazid on the ΔC-D ratio values of sensitive strains and clinical isolates are different. In the rifampicin resistance test, after incubating for 24 h, when the drug concentration is 16 mg / L, there are significant differences in the ΔC-D ratio between the sensitive strain L0 and the isolate L2 (P<0.05); in the isoniazid resistance test, when the drug concentration is 4 mg / L, there are significant differences in the ΔC-D ratio between the sensitive strain L0 and the isolate L4 (P<0.05). When the ΔC-D ratio is greater than the critical value of -0.02, it is defined as a drug-resistant strain.

[0114] In the above embodiments, the method for preparing a Mycobacterium tuberculosis sample includes:

[0115] The first step, material preparation:

[0116] Preparation of 7H9 medium: Take 4.7 g of 7H9 powder and 90 mL of OADC, dissolve them in 900 mL of ddH2O, and sterilize at 121 °C for 15 min;

[0117] Preparation of bacterial suspension: Centrifuge the Mycobacterium tuberculosis sensitive strain (L0) and clinical isolates (rifampicin-resistant strain L2, isoniazid-resistant strain L4) resuscitated with 7H9 medium, and use a dropper to suck the colonies at the bottom of the tube into injection water, and adjust the McFarland turbidity to 1 with a bacterial ultrasonic dispersion counter.

[0118] The second step, establishing a drug incubation system for sensitive strains and drug-resistant strains:

[0119] The bacterial suspension with a McFarland turbidity of 1 was aliquoted into 2 mL per portion, centrifuged at 3000 g for 20 min, and the supernatant was discarded. The precipitate was transferred to incubation systems containing 7H9 medium, heavy water, ddH2O, and drugs. The content of heavy water in the system was 50% (volume ratio), and the final concentrations of the drugs were: isoniazid 0 mg / L, 4 mg / L, 16 mg / L, 64 mg / L; rifampicin 0 mg / L, 16 mg / L, 64 mg / L, 256 mg / L. A drug resistance incubation system for isoniazid / rifampicin was established, with 3 replicates for each system. Incubation was carried out in a constant temperature incubator at 37°C. Samples were taken after 24 h for Raman detection.

[0120] The third step is the treatment of the sample to be tested, including:

[0121] Inactivation;

[0122] Specifically, for the inactivation method, take 1.0 mL of the incubation system in a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min, discard the supernatant, resuspend with the prepared cryopreservation solution, and inactivate in a metal bath at 80°C for 30 min. If the sample to be tested cannot be detected immediately, the inactivated sample is stored at -80°C.

[0123] Slide preparation;

[0124] Specifically, for the inactivated sample to be tested, centrifuge at 10000 rpm for 2 min, discard the supernatant, resuspend with 500 μL of ddH2O, centrifuge at 10000 rpm for 2 min, remove the supernatant, repeat the washing 2 - 3 times, and finally resuspend with ddH2O to a final concentration of 10 6 CFU / mL. Take 10 μL of the resuspended solution and transfer it to the detection substrate CaF2 glass slide. Three parallel spot samplings are performed for each sample, and it is air-dried naturally in a biosafety cabinet for testing.

[0125] In the detection method of the above embodiments, by using Raman technology to automatically detect clinical pathogenic microbial plaques, the detection of each drug resistance of pathogenic bacteria per sample can be quickly achieved within 0.5 - 1 minute.

[0126] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An automated rapid detection instrument for microbial Raman drug resistance, characterized in that, It includes an electric displacement platform, an imaging module, an excitation light module, a micro-focusing module, a Raman main optical path and transmission module, a coaxial illumination module, and an automatic acquisition and control module; The electric displacement platform is used to place the sample chip; The imaging module is used to capture the panoramic information of the sample chip and the microscopic information of the dental plaque, and cooperate with the electric translation platform to quickly locate the dental plaque to be measured on the sample chip, and determine the collection position of the dental plaque to be measured; The excitation light module is used to emit laser light; The micro-focusing module is used to automatically focus the laser light on the dental plaque to be measured at the collection position, so that the dental plaque to be measured generates Raman signals; The coaxial illumination module is used to provide coaxial illumination light for the micro-focusing module; The Raman main optical path and transmission module are used to obtain the Raman spectrum of the dental plaque to be measured; The automatic acquisition and control module is used to control the excitation light module, the Raman main optical path and transmission module, the micro-focusing module, the coaxial illumination module, the imaging module and the electric displacement platform, and process the Raman spectrum of the collected dental plaque to be measured to realize the automatic detection of the dental plaque to be measured; The excitation light module includes a laser, an electric shutter, a beam expander, and an electrically adjustable attenuator; the Raman main optical path and transmission module include a broadband mirror, a dichroic mirror, a second lens, a pinhole, a microscope objective, a spectrometer, and a detector; The laser light emitted by the laser passes through the electric shutter, the beam expander, the electrically adjustable attenuator, the mirror, the dichroic mirror, and the broadband mirror in sequence and is reflected to the microscope objective to be focused on the sample chip on the electric displacement platform; The Raman signal light generated by the excitation of the sample chip passes through the microscope objective, the broadband mirror, the dichroic mirror, the second lens, the pinhole, and the spectrometer in sequence and is emitted to the detector; The coaxial illumination module includes an LED light source and a semi-transparent and semi-reflective mirror, and the imaging module includes a CCD camera and a first lens; The white light emitted by the LED light source passes through the semi-transparent and semi-reflective mirror and the microscope objective in sequence and is focused on the sample chip; the reflected light formed by the LED light source irradiating the sample chip enters the CCD camera through the microscope objective, the semi-transparent and semi-reflective mirror, and the first lens to form an image of the sample chip; The excitation light module, the Raman main optical path and transmission module, the coaxial illumination module, and the imaging module are fixed inside the same optical box body. The optical box body is a fully enclosed structure, and the Raman main optical path and transmission module and the micro-focusing module are located in the same plane.

2. The automated rapid microbial Raman drug resistance detection instrument according to claim 1, characterized in that, The automatic microbial Raman drug resistance rapid detection instrument includes an image observation mode and a Raman measurement mode; The image acquisition mode is used to obtain the image information of the sample chip; The Raman measurement mode is used to realize the acquisition of the Raman information of the dental plaque to be measured on the sample chip.

3. The automated rapid microbial Raman drug resistance detection instrument according to claim 1, characterized in that, The bandwidth range of the broadband mirror is from the excitation wavelength of the laser plus 100 nm to 200 nm.

4. The automated rapid microbial Raman drug resistance detection instrument according to claim 1, characterized in that, The detection instrument also includes a sterilization module, which is used to eliminate microorganisms in the environment and prevent cross-contamination.

5. The detection method of the automated microbial Raman drug resistance rapid detection instrument according to any one of claims 1-4, characterized in that, It includes the following steps: The imaging module captures the image of the entire sample chip to obtain the position information of each dental plaque to be measured; Move the mobile electric displacement platform to the first plaque to be measured and determine the best acquisition position of the plaque to be measured; The excitation module emits laser, and the laser is automatically focused on the plaque to be measured through the microscopic focusing module, so that the plaque to be measured generates Raman signals; The Raman main optical path and transmission module acquires the Raman spectrum of the plaque, and completes the plaque detection through the automatic acquisition control module; Move to the next plaque to be measured on the electric displacement platform and repeat the above process. Process the Raman spectrum data of each plaque to be measured to complete the drug resistance detection.

6. The detection method of the automated microbial Raman drug resistance rapid detection instrument according to claim 5, characterized in that, Determine the best acquisition position of the plaque to be measured, including: Acquire the plaque image; Process the image to convert it into a grayscale image and perform filtering; Judge the image pixel points. If it is higher than the set threshold, the image pixel point is a bright spot; if it is lower than the set threshold, check whether there is a plaque at its surrounding position; Locate the dense area of bright spots as the position of the plaque.

7. The detection method of the automated microbial Raman drug resistance rapid detection instrument according to claim 5, characterized in that, The automatic focusing process includes: Use the hill climbing search algorithm to select multiple position points of the first plaque to be measured; Use the clarity evaluation function value of the multiple position points to fit the local curve where the multiple position points are located; Judge whether there is a peak greater than the set threshold in the curve. If there is such a peak, return the point before the point closest to the maximum value among the position points of this section. Take this point as the starting point and the point after the maximum value point as the end point, re-divide the search range and reduce the search step size; repeat the search until the required focusing accuracy is achieved. If there is no such peak, continue to move in the original direction and repeat the above focusing operation to finally confirm the optimal focal plane.

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