CN109266717B - Method and device for detecting bacterial drug resistance through single cell analysis - Google Patents
Method and device for detecting bacterial drug resistance through single cell analysis Download PDFInfo
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Abstract
A method for detecting the drug resistance of bacteria by single cell analysis includes such steps as mixing a certain quantity of bacteria sample with a certain quantity of diluted antibiotic solution, putting it on a slide glass, covering it with a cover glass, and sealing by sealant to form a plate-type biochemical reaction system in which bacteria and diluted antibiotic solution react. The bacteria are observed and positioned by planar scanning through a phase contrast microscope, and then the positioned bacteria are scanned by laser to obtain a Raman spectrum, so that the drug resistance condition of the bacteria can be obtained through analysis. The invention also provides a device for realizing the method, which comprises a plate-type micro-biochemical reaction system, a phase contrast microscope, a high-power optical microscope, a Raman spectrum excitation system and a Raman spectrum collection system. And (3) observing and positioning the bacteria through a phase contrast microscope and a high-power optical microscope, and then acquiring the change of the Raman spectrum of the bacteria by using a Raman spectrum excitation system and a Raman spectrum collection system so as to detect the drug resistance of the bacteria.
Description
Technical Field
The invention relates to the field of physics, in particular to an optical detection technology, and particularly relates to a method and a device for detecting bacterial drug resistance through single cell analysis.
Background
According to comprehensive analysis of a plurality of data by professors related to the medical college of Zhejiang university, the total usage of antibiotics in China is about 15-20 ten thousand tons every year, and the usage accounts for about 50% of the total usage in the world. Meanwhile, the antibiotic consumption and the antibiotic discharge density in the eastern China basin are 6 times higher than those in the western China basin due to the influence of dense population, pharmaceutical industry, agriculture, breeding industry distribution and other factors. The large scale, often unnecessary, use of antibiotics means that more and more pathogens are not susceptible to drugs and previously treatable infections may be life threatening.
The extensive existence of antibiotic residues in the environment and the spread and diffusion of bacterial drug resistance are caused by the massive use of antibiotics, so that the ecological environment and the human health are further influenced, and the problems of antibiotic environmental pollution and bacterial drug resistance are more and more widely concerned by the society. In 2016, 8/26/8/14 departments of national institutes of health, Oncology of health, Inc., in "action plan for suppressing bacteria resistance in China (2016-2020): the method comprises the steps of strengthening the antibacterial drug application and the construction of a drug resistance control system and perfecting the antibacterial drug application and a bacterial drug resistance monitoring system. The national health committee in march 2018 reissues a notice about continuously doing relevant work of antibacterial clinical application management, emphasizes further strengthening the antibacterial clinical application management, limits the use of antibiotics to be upgraded again, and prevents the situations of 'no medicine available' and 'no strategy' aiming at bacteria.
At present, most clinical diagnosis does not carry out pathogen fine detection, and especially when some serious infections such as septicemia are diagnosed and treated, life risks are often caused by the fact that the medicine cannot be taken quickly and accurately. In order to better serve the clinical purpose, the fine medication has very important significance. Not only can greatly reduce the use amount of antibiotics, but also saves resources and protects the environment. Can effectively and accurately treat diseases and save lives of patients in time, and has great social significance.
The prior art methods for detecting bacterial drug resistance include:
1: conventional identification methods
Mainly by the morphological, biochemical, immunological examination of the pathogen. Because the number of each pathogen is small, the identification of physiology, biochemistry and immunology is not facilitated, and the identification is carried out after the number of the pathogens reaches a certain degree through in vitro culture. Traditional pathogen detection methods are mostly specific, culturable bacteria and take a long time. The detection time of about 2 days is needed for culturable bacteria, about 7 days is needed for strains with slow growth, and the detection time is useless for bacteria which cannot be cultured in a laboratory. Furthermore, conventional methods have difficulty distinguishing whether a infection is caused by one or more bacterial pathogens. Normally, 2-3 days are required from pathogen preparation to the final result. These methods have the advantage of accuracy and the disadvantages of time and labor consumption and the tendency of the sample to be contaminated.
2: automated instrumentation detection
According to the difference of different biological characters and metabolites of pathogens, a trace rapid culture medium and a trace biochemical reaction system are gradually developed, so that the original slow and tedious manual operation becomes automatic. A variety of automatic pathogen identification and susceptibility testing systems are known, such as VITEK-AutoMicrobicSystem (AMS), PHOENIXTM, MicroScan, sensitre, ABBott (MS-2System), AUTOBACIDXSys-tert, and others. The automatic systems have advanced microcomputer systems and wide identification functions, are suitable for clinical microbiological laboratories, epidemic prevention and commercial inspection systems, mainly have the functions of bacteria identification, bacteria drug susceptibility tests, Minimum Inhibitory Concentration (MIC) measurement and the like, and have greatly improved accuracy and reliability.
Although these devices are used in some locations, there are some disadvantages: (1): they are only suitable for common pathogens that can be cultured in vitro, which still takes a long time for the sample to be cultured in vitro; (2): the species of microorganisms can be known only from one side, and some microorganisms exhibiting unique biochemical characteristics cannot be correctly discriminated.
3: molecular biological identification method
Several new molecular identification methods are now being developed to identify pathogens based on analysis of high throughput genetic sequencing data and biomolecular mass spectrometry.
(1) Gene sequencing analysis
The genetic basis of the drug resistance of bacteria is gene mutation or the acquisition of drug resistance genes, and the targeted detection of the gene mutation or the related drug resistance genes is an important means and way for detecting and monitoring the drug resistance of the bacteria. At present, the latest generation gene sequencer manufacturers mainly comprise Illumina, Roche, ABI and the like. Illumina upgrades Genome Analyzer II to Genome Analyzer IIx, and realizes the ambitious goal of obtaining 95GB data in a single operation. The Roche diagnostics introduced the Genome sequence FLX System (GS FLX) as a second generation Genome sequencing System with better performance. The flux of GS FLX is increased by 5 times at a time, and the accuracy and the reading length are further improved. ABI introduced a new generation sequencing platform for SOLiD 3. The method is characterized in that the method is based on the continuous connection and synthesis of four-color fluorescence labeling oligonucleotides, replaces the traditional polymerase connection reaction, and can carry out large-scale amplification and high-throughput parallel sequencing on single-copy DNA fragments. These instruments are expensive and are essentially monopolized by foreign instrument factories.
The gene sequencing method has the advantages that the bacteria identification can be carried out on the molecular level, various limitations of the traditional culture method are overcome, the identification of rare bacteria, slow-growing bacteria, uncultured bacteria and the like is particularly important, and the analysis of data is more accurate. The method has the defects that the gene sequencing method is mostly based on 16S rRNA gene to carry out bacterial identification and classification, certain similar species are difficult to distinguish, the interpretation of the sequencing result is complex, the speed is slow and the cost is high. The whole inspection process needs very fine operation and takes very long time. The reagents for molecular biological tests are expensive, and because of the small amount of specimens, the specimens are often collected and tested together, so that the cost is saved. In addition, it is feasible that gene sequencing techniques identify drug resistance genes to indirectly infer pathogen resistance. However, this technique relies on a reference sequence of known drug-resistant genes, and cannot analyze unknown drug resistance, and there is often a difference between genotypic and phenotypic resistance. Even the several current U.S. FDA-approved gene diagnostic methods must be used under stringent restriction conditions.
(2) Mass spectrometry method
The principle of the mass spectrometry is that biomolecules are ionized, ions accelerate to fly through a flight pipeline under the action of an electric field, protein fingerprint spectrums are formed according to different flight times of arriving at a detector, and then the protein fingerprint spectrums are compared with standard fingerprint spectrums in a database through software, so that the aim of identification is fulfilled. The technology is initially applied to strain identification, has the characteristics of high sensitivity, rapidness and accuracy, and is gradually applied to bacterial drug resistance detection and drug resistance mechanism research. The MALDI-TOF MS technology is mainly used for detecting the existence condition of drug-resistant related enzymes of bacteria by detecting the modification and hydrolysis conditions of antibacterial drugs, or detecting the change of fingerprint spectrums of drug-resistant strains and sensitive strains to judge the drug-resistant strains and the sensitive strains, thereby achieving the purpose of detecting the drug resistance of bacteria. Mass spectrometers for microbial identification include Vitek-MS, Merrier, France, and Biotyper, Bruker, Germany. Vitek-MS was originally obtained from a mass spectrometer Axima, introduced by Shimadzu, Japan, whose microbial database SARAMIS was developed by Anagnos Tec, Germany. The Biotyper system is a product independently developed by bruker. These instruments are expensive and are essentially monopolized by foreign instrument factories.
However, this technique still has some drawbacks in bacterial identification. Since the theoretical support of mass spectrometry is to analyze the spectra obtained from the mass-to-charge ratio of the particles after ionization of the sample, the reliability depends on the uniqueness of the mass-to-charge ratio. However, the same bacteria, depending on the culture conditions or the chemical extraction method, may produce different mass spectra, resulting in errors in the analysis results. Even if the above conditions are strictly controlled, mass spectrometry cannot completely accurately determine the bacterial species. In addition, compared with the traditional drug sensitivity test, the MALDI-TOF MS technology can only distinguish sensitive strains from drug-resistant strains, and cannot obtain accurate MIC values, and the technology needs to firstly utilize known sensitive strains and known drug-resistant strains for modeling when judging the drug-resistant strains and the sensitive strains. At present, the technical equipment and the matched reagent are expensive, the detection of drug resistance types is limited, and the wide-range popularization and application of the technology in clinic and basic level are restricted.
Disclosure of Invention
The invention aims to provide a method and a device for detecting bacterial drug resistance through single cell analysis, and the method and the device for detecting bacterial drug resistance through single cell analysis are used for solving the technical problems of long time consumption, complex sample preparation method and easy interference in the prior art for detecting bacterial drug resistance.
The invention provides a method for detecting bacterial drug resistance through single cell analysis, which comprises the following steps:
1) obtaining a bacterial sample from a body fluid known to contain or likely to contain bacterial cells by centrifugation or filtration or concentration or precipitation or extraction;
2) dripping a bacterial sample and an antibiotic diluent on a glass slide at the same time or step by step, covering by using a cover glass and sealing by using a sealant at four sides to form a plate-type micro-biochemical reaction system in which bacteria and the antibiotic diluent are subjected to biochemical reaction;
3) after the biochemical reaction is completed, the plate-type micro-biochemical reaction system is arranged on an objective table, the objective table is driven to move by a motor, a phase contrast microscope is adopted for carrying out plane scanning, and bacteria are observed and positioned;
4) the motor is utilized to drive the objective table to move, bacteria needing to be analyzed in the plate-type micro-biochemical reaction system are positioned in a high-power optical microscope field of view, then the bacteria are further positioned through the high-power optical microscope, the positioned bacteria in the laser scanning field of view are guided to carry out Raman spectrum imaging, and the bacteria needing to be analyzed in the microscope field of view are completed one by one;
5) and (4) driving the objective table to move by using a motor, repeating the step (4), completing Raman spectrum imaging of other bacteria needing to be analyzed in the plate-type micro-biochemical reaction system, and finally obtaining a bacterial drug resistance result through data analysis.
Further, the thickness of the slide glass of the plate-type micro biochemical reaction system is less than 2.5 mm, the thickness of the cover glass is less than 2.0 mm, and the distance between the slide glass and the cover glass is between 1 micron and 5mm, preferably between 5 microns and 10 microns.
Furthermore, the sealant adopts Vaseline, paraffin, adhesive tape, sealing film or grease or sealant.
Further, the Raman spectrum scanning imaging resolution is less than 20 microns.
The invention also provides a device for realizing the method, which comprises a plate-type micro-biochemical reaction system, wherein the plate-type micro-biochemical reaction system comprises a glass slide and a cover glass, the cover glass is arranged on the upper side of the glass slide, the plate-type micro-biochemical reaction system is arranged on an object stage, and the object stage is connected with a motor;
the Raman spectrum excitation system is sequentially provided with a Raman spectrum excitation light source, a first light beam shaper, a laser scanning device, a second light beam shaper, a third beam splitter, a second beam splitter and an objective lens along the laser irradiation direction;
the Raman spectrum collection system is sequentially provided with an objective lens, a second beam splitter, an optical filter, a coupling lens and a spectrometer along the direction of back Raman scattered light collection light, and the spectrometer, the coupling lens, the optical filter, the second beam splitter and the objective lens are arranged on a coaxial light path;
the phase difference microscope system is sequentially provided with a first illumination light source, an annular diaphragm, a condenser, a phase objective, a first beam splitter, a first imaging lens and a first CCD camera in the imaging direction, wherein the first illumination light source, the annular diaphragm and the phase objective are arranged on a coaxial light path, and the first imaging lens and the first CCD camera are arranged on a 45-degree light path of the first beam splitter;
the device also comprises a high power optical microscope system, wherein a second imaging lens and a second CCD camera are arranged at the lower end of the third beam splitter, a second illuminating light source is arranged at the upper end of the plate type micro-biochemical reaction system, the second illuminating light source, the objective lens, the second beam splitter, the third beam splitter, the second imaging lens and the second CCD camera form the high power optical microscope system, and the second beam splitter, the objective lens and the second illuminating light source are arranged on a central axis.
Further, the phase microscope phase objective has a magnification of 20-100 times, preferably 40 times;
further, the magnification of the high-power optical microscope objective lens is between 40 and 100 times, and preferably 60 times;
furthermore, the laser focusing lens, the Raman scattering light collecting lens and the optical imaging lens in the Raman spectrum excitation system, the Raman spectrum collection system and the high-power optical microscope system adopt the same objective lens.
Furthermore, the coupling mirror is composed of at least one lens, can eliminate imaging phase difference and is used for focusing the Raman scattering light into the spectrometer.
Furthermore, the periphery of the space formed by the cover glass and the glass slide is sealed by sealant.
Further, the laser scanning device is connected with a beam shaping system, and the laser scanning device and the second beam shaper are arranged on a 45-degree light path of the second beam splitter.
Furthermore, the objective lens, the second beam splitter, the third beam splitter, the second imaging mirror and the second CCD camera are arranged at the lower end of the plate-type micro-biochemical reaction system.
Furthermore, the first illumination light source, the annular diaphragm and the condenser are arranged at the upper end of the plate-type micro-biochemical reaction system, and the phase objective lens, the first beam splitter, the first imaging lens and the first CCD camera are arranged at the lower end of the plate-type micro-biochemical reaction system.
Imaging principle of phase contrast microscopy: when the light passes through the transparent ring of the ring-shaped diaphragm in microscopic examination, the light is condensed into a light beam by the condenser, and when the light beam passes through the cell to be examined, the light beam is diffracted to different degrees due to different optical paths of all parts. The image formed by the transparent ring just falls on the rear focal plane of the objective lens and coincides with the conjugate plane on the phase plate. Therefore, the direct light that is not deflected passes through the conjugate plane, and the diffracted light that is deflected passes through the compensation plane. Because the conjugate surface and the compensation surface on the phase plate have different properties, the light rays passing through the two parts respectively generate certain phase difference and intensity reduction, and the two groups of light rays are converged by the rear lens and then travel on the same light path, so that the direct light and the diffracted light generate light interference, and the phase difference is changed into amplitude difference. Thus, when the phase difference is detected by a phase difference microscope, the phase difference which can not be distinguished by human eyes is converted into the light and shade difference which can be distinguished by human eyes through the light rays of the colorless transparent body.
The principle of Raman spectrum analysis is as follows: after the medicine and the pathogen cell have biochemical action, the molecular structure of the pathogen cell can change, a reaction is immediately performed on the Raman spectrum of the pathogen, the change of the molecular structure of the pathogen cell with drug resistance to the medicine can be avoided, and the spectrum of the pathogen cell is maintained unchanged. Thereby rapidly reflecting the resistance of pathogens to the applied drugs. Meanwhile, the method can obtain the concentration of the medicine which can completely inhibit the growth of the pathogen, and know the accurate medicine application of doctors.
The method for detecting bacterial drug resistance by single cell analysis is characterized by that a certain quantity of bacterial sample and a certain quantity of antibiotic diluent are mixed and placed on a glass slide, covered by a cover glass and sealed by means of sealant to form a plate-type micro-biochemical reaction system in which the bacteria and antibiotic diluent can produce biochemical reaction. The bacteria are observed and positioned by planar scanning through a phase contrast microscope, the bacteria to be measured are further positioned through a high-power optical microscope, the change of the Raman spectrum of the bacteria is obtained by scanning the positioned bacteria through laser, and the drug resistance condition of the bacteria can be obtained by analyzing the change of the Raman spectrum of the bacteria. The invention has simple sample processing and high measuring speed, and is the best choice for replacing the prior art in the future.
Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The bacteria drug resistance Raman spectrum detection technology can realize the applications of bacteria rapid identification and identification, drug sensitivity test and the like. Moreover, the technology is also widely used in the field of drug resistance detection of fungi, chlamydia, viruses and the like. Through the subsequent gradual completion of the research and development and the achievement transformation of related products, the clinical rapid and on-site requirements of pathogens in the precise medical field can be met. Provides a powerful tool for doctors, diagnoses diseases more quickly in clinical treatment decision, uses antibiotics more accurately and realizes efficient, safe and accurate treatment. The invention uses phase contrast microscopy to locate unstained living cells. The phase contrast microscope is based on the difference of refractive index and thickness of each part of cell, and the light ray generates interference phenomenon when passing through the cell, so that the original transparent cell shows obvious light and shade difference, and living cells and some fine structures in the cell which can not be seen or can not be seen clearly under the common optical microscope and the dark field microscope can be observed clearly. And the change of the biomolecule cells is dynamically monitored in real time through Raman spectrum to research the cellular reaction of diseases, drugs or toxins. And determining the drug sensitivity characteristics of the bacteria. The invention has the advantages of extremely low sample consumption, non-invasiveness and the like, so that the invention has wide application in analysis of bacteria, fungi, viruses and chlamydia, screening of drug sensitivity and the like.
Drawings
FIG. 1 is a schematic structural diagram of a device for detecting bacterial drug resistance by single cell analysis according to the present invention. The part names are as follows:
11: a first illumination light source; 12: an annular diaphragm; 13: a condenser; 14: an object stage; 15: a phase objective lens; 16: first beam splitter 17: a first imaging mirror; 18, a first CCD camera; 21: a spectrometer; 22: a coupling mirror; 23: an optical filter; 24: a second beam splitter; 25: an objective lens; 26: a bacterial sample; 27: a second illumination light source; 31: a second CCD camera; 32: a second imaging mirror; 33: a third beam splitter; 41: a Raman spectrum excitation light source; 42: a first beam shaper; 43: a laser scanning device; 44: a second beam shaper; 51: a cover glass; 52: a sealant; 53: a glass slide; 54: an antibiotic diluent; 61: an electric motor.
FIG. 2 is a 40 times objective optical microscope photograph of E.coli.
Detailed Description
Example 1
As shown in FIG. 1, the present invention provides an apparatus for detecting bacterial drug resistance by single cell analysis, comprising a plate-type micro-biochemical reaction system, wherein the plate-type micro-biochemical reaction system is provided with a glass carrying plate 53 and a cover glass 51, and is sealed by a sealant 52, the plate-type micro-biochemical reaction system is arranged on a stage 14, and a motor 61 is connected with the stage 14 to drive the stage 14 to move relatively.
The Raman spectrum excitation system is further provided, and a Raman spectrum excitation light source 41, a first beam shaper 42, a laser scanning device 43, a second beam shaper 44, a third beam splitter 33, a second beam splitter 24 and an objective lens 25 are sequentially arranged in the laser irradiation direction in the Raman spectrum excitation system.
Specifically, the laser scanning device 43 is connected to a second beam shaper 44, and the laser scanning device 43 and the second beam shaper 44 are disposed on the 45-degree optical path of the second beam splitter 24.
The Raman scattering optical system is characterized by further comprising a Raman spectrum collecting system, wherein an objective lens 25, a second beam splitter 24, an optical filter 23, a coupling lens 22 and the spectrometer 21 are sequentially arranged in the Raman spectrum collecting system along the direction back to the Raman scattering light collecting light, and the spectrometer 21, the coupling lens 22, the optical filter 23 and the objective lens 25 are arranged on a coaxial light path.
The phase difference microscope system is sequentially provided with a first illumination light source 11, an annular diaphragm 12, a condenser 13, a phase objective 15, a first beam splitter 16, a first imaging lens 17 and a first CCD camera 18 in the imaging direction, wherein the first illumination light source 11, the annular diaphragm 12 and the phase objective 15 are arranged on a coaxial light path, and the first imaging lens 17 and the first CCD camera 18 are arranged on a 45-degree light path of the first beam splitter 16;
specifically, the first illumination light source 11, the annular diaphragm 12 and the condenser 13 are arranged at the upper end of the plate-type micro-biochemical reaction system, and the phase objective 15, the first beam splitter 16, the first imaging mirror 17 and the first CCD camera 18 are arranged at the lower end of the plate-type micro-biochemical reaction system.
The high-power optical microscope system is arranged by adopting an inverted microscope, a second illumination light source 27, an objective lens 25, a second beam splitter 24, a third beam splitter 33, a second imaging lens 32 and a second CCD camera 31 are sequentially arranged in the imaging direction of the high-power optical microscope, the lower end of the third beam splitter 33 is provided with the second imaging lens 32 and the second CCD camera 31, and the second beam splitter 24, the objective lens 25 and the second illumination light source 27 are arranged on a central axis;
specifically, the second illumination light source 27 is disposed at the upper end of the plate-type biochemical reaction system, and the objective lens 25, the second beam splitter 24, the third beam splitter 33, the second imaging mirror 32 and the second CCD camera 31 are disposed at the lower end of the plate-type biochemical reaction system.
The process of observing and locating bacteria of the present invention is: the objective table 14 is driven by the motor 61 to move, and the phase contrast microscope system is used for scanning the plate type micro-biochemical reaction system to observe and position bacteria. After the bacteria needing to measure the raman spectrum are positioned, the motor 61 is used for driving the objective table 14 to move so as to position the bacteria needing to be analyzed in the field of view of the high-power optical microscope, the second illumination light source 27 is used for illuminating the bacteria sample 26, and then the bacteria sample is amplified and imaged to the second CCD camera 31 through the objective lens 25 and the second imaging lens 32, so that the bacteria sample 26 is further positioned.
The Raman spectrum scanning imaging process of the invention comprises the following steps: firstly, a raman spectrum excitation light source 41 is used for emitting laser, the laser is shaped by a first beam shaper 42, then passes through a laser scanning device 43, a second beam shaper 44 and a third beam splitter 33, is reflected by a second beam splitter 24, and is finally focused on a bacterial cell sample 26 through an objective lens 25 to generate raman scattering light, and a part of the back raman scattering light returns through an objective lens 25 collimation original light path and is focused into a spectrometer 21 through the second beam splitter 24, a filter 23 and a coupling mirror 22 for cell raman spectrum measurement. The laser emitted from the raman spectrum excitation light source 41 is scanned by the laser scanning device 43 to collect a raman spectrum image of the entire bacterial cell. The raman spectroscopy excitation light source 41 uses a 1064nm laser excitation source to focus a total laser intensity of about 70mW onto the cell sample through a 60-fold/NA 0.95 objective lens 25. The entire bacterial cell was scanned using 0.7 μm steps with an entire cell raman spectrum integration time of 3 seconds.
Specifically, the first beam shaper 42 can change the laser with transverse gaussian light intensity distribution into the laser with transverse uniform light intensity distribution, so as to avoid the cell damage caused by the over-strong central laser intensity during laser focusing.
Specifically, the second beam shaper 44 is a 2.0-fold beam expander.
Specifically, the coupling mirror 22 is an aspheric lens with a focal length of 15mm and a diameter of 20 mm.
The process of analyzing and detecting the drug resistance of the bacteria comprises the following steps: firstly, blood of a patient is obtained, bacteria-containing concentrated solution is obtained through centrifugal separation, filtering by a 0.25um filter element and centrifugal concentration at 650 revolutions per minute, 350 microliters of ciprofloxacin diluent 54 with a certain concentration is dripped on a glass slide (1.2mm, quartz glass) by using a micro syringe, and then 300 microliters of bacteria-containing concentrated solution is dripped into the ciprofloxacin diluent 54. Covered with a cover glass (0.5mm, quartz glass). Sealing with vaseline to form a plate-type micro-biochemical reaction system. The cells were then incubated at 37 ℃ in an incubator in which the bacteria and antibiotics reacted biochemically.
After 60 minutes, raman spectroscopy was started. The samples were first scanned using a phase contrast microscope system to observe and locate the bacteria. The first illumination light source 11 emits illumination light, and the light transmitted through the condenser 13 is formed into a hollow cone of light by the annular diaphragm 12 and the condenser 13, and is focused on the cell sample 26. The difference of the refractive index and the thickness of each part of the cell generates an interference phenomenon when light passes through the cell, so that the original transparent cell shows obvious light and shade difference. By using the phase objective 15 and the first imaging lens 17, the cell sample 26 is imaged on the first CCD camera 18, so that living cells and some fine structures in the cells which cannot be seen or cannot be seen clearly under a common optical microscope and a dark-field microscope can be observed clearly.
After the bacteria needing to measure the raman spectrum are positioned, the motor 61 is used for driving the objective table 14 to move, the bacteria needing to be analyzed are positioned in the field of view of the high-power optical microscope, then the bacteria are further positioned through the high-power optical microscope, the bacteria sample 26 is illuminated by the second illumination light source 27, and then the bacteria are magnified and imaged to the second CCD camera 31 through the objective lens 25 and the second imaging lens 32. FIG. 2 shows optical microscopy imaging of E.coli using a 40 x objective. And finally, guiding laser to scan the bacteria to be detected positioned in the high-power optical microscope field of view for Raman spectrum imaging. The Raman spectrum excitation light source 41 is used for emitting laser, the laser is shaped by the first shaper 42, then passes through the laser scanning device 43, the second beam shaping system 44, the third beam splitter 33, then is reflected by the second beam splitter 24, and finally is focused on the bacterial cell sample 26 through the objective lens 25 to generate Raman scattered light, and a part of the back Raman scattered light returns through the objective lens 25 and is collimated on the original light path and focused into the spectrometer 21 through the second beam splitter 24, the optical filter 23 and the coupling mirror 22 to be used for cell Raman spectrum measurement. The laser emitted from the raman spectrum excitation light source 41 is scanned by the laser scanning device 43 to collect a raman spectrum image of the entire bacterial cell. The motor 61 is used for driving the objective table 14 to move, so that other bacteria to be analyzed are positioned in the high-power optical microscope field, and the bacteria to be analyzed in each high-power optical microscope field are completed one by one. And finally, obtaining the bacterial drug resistance result through classification statistics, data averaging and Raman spectrum analysis.
Results of measurement are shown in the following table, and table 1 is a comparison of MIC measurements for the effect of the present invention with escherichia coli and ciprofloxacin. The MIC values determined were in perfect agreement with the MICs obtained by standard methods.
Note: BMD: traditional broth microdilution; E-TEST, U.S. fly antibiotic sensitive TEST strips; VITEK-2, a French Meiriee automatic identification and drug sensitivity test system; measuring time: VITEK-2 and E-TEST, 16-24 hours; raman: for 90 minutes.
The Raman spectrum can be used for identifying the drug resistance of bacteria on a molecular level, and particularly has an unparalleled advantage on identification of rare bacteria, slow-growing bacteria, uncultured bacteria and the like. In particular, the mixed bacteria can be analyzed for each bacterial species and distribution.
Although the preferred embodiments of the present invention have been described, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.
Claims (9)
1. A device for detecting bacterial drug resistance through single cell analysis is characterized by comprising a plate-type micro-biochemical reaction system, wherein the plate-type micro-biochemical reaction system comprises a glass slide and a cover glass, the cover glass is arranged on the upper side of the glass slide, the plate-type micro-biochemical reaction system is arranged on an object stage, and the object stage is connected with a motor; the Raman spectrum excitation system is sequentially provided with a Raman spectrum excitation light source, a first light beam shaper, a laser scanning device, a second light beam shaper, a third beam splitter, a second beam splitter and an objective lens along the laser irradiation direction; the Raman spectrum collection system is sequentially provided with an objective lens, a second beam splitter, an optical filter, a coupling lens and a spectrometer along the direction of back Raman scattered light collection light, and the spectrometer, the coupling lens, the optical filter, the second beam splitter and the objective lens are arranged on a coaxial light path; the phase difference microscope system is sequentially provided with a first illumination light source, an annular diaphragm, a condenser, a phase objective, a first beam splitter, a first imaging lens and a first CCD camera in the imaging direction, wherein the first illumination light source, the annular diaphragm and the phase objective are arranged on a coaxial light path, and the first imaging lens and the first CCD camera are arranged on a 45-degree light path of the first beam splitter; the device also comprises a high power optical microscope system, wherein a second imaging lens and a second CCD camera are arranged at the lower end of the third beam splitter, a second illuminating light source is arranged at the upper end of the plate type micro-biochemical reaction system, the second illuminating light source, the objective lens, the second beam splitter, the third beam splitter, the second imaging lens and the second CCD camera form the high power optical microscope system, and the second beam splitter, the objective lens and the second illuminating light source are arranged on a central axis.
2. The apparatus for detecting bacterial resistance according to claim 1, wherein the phase microscope phase objective has a magnification of 20-100 times.
3. The apparatus for detecting bacterial resistance according to claim 1, wherein the objective lens magnification of the high power optical microscope is between 40-100 times.
4. The apparatus for detecting bacterial resistance through single cell analysis according to claim 1, wherein the laser focusing lens, the raman scattering light collecting lens and the optical imaging lens in the raman spectroscopy excitation system, the raman spectroscopy collection system and the high power optical microscope system use the same objective lens.
5. The apparatus of claim 1, wherein the coupling mirror comprises at least one lens for focusing raman scattered light into the spectrometer.
6. The apparatus for detecting bacterial resistance through single cell analysis according to claim 1, wherein the space formed by the cover glass and the slide glass is sealed with sealant.
7. The apparatus of claim 1 wherein the laser scanning device and the second beam shaper are disposed in a 45 degree optical path of the second beam splitter.
8. The apparatus for detecting bacterial drug resistance through single cell analysis according to claim 1, wherein the objective lens, the second beam splitter, the third beam splitter, the second imaging lens and the second CCD camera are disposed at the lower end of the plate-type micro biochemical reaction system.
9. The apparatus of claim 1, wherein the first illumination source, the annular diaphragm, and the condenser are disposed at the upper end of the plate-type biochemical reaction system, and the phase objective, the first beam splitter, the first imaging mirror, and the first CCD camera are disposed at the lower end of the plate-type biochemical reaction system.
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| KR20210052389A (en) | 2018-08-27 | 2021-05-10 | 리제너론 파마슈티칼스 인코포레이티드 | Use of Raman spectroscopy in downstream purification |
| CN110231330A (en) * | 2019-05-31 | 2019-09-13 | 南方科技大学 | A kind of method of single cell Raman spectrum technology measurement ammoxidation archaeal metabolic activity |
| CN110220883B (en) * | 2019-06-26 | 2023-02-28 | 中国科学院长春应用化学研究所 | Single cell control device |
| GB201919190D0 (en) * | 2019-12-23 | 2020-02-05 | Celltool Gmbh | Identification of microbial contaminations or infections in liquid samples by raman spectroscopy |
| CN113234789A (en) * | 2021-06-07 | 2021-08-10 | 东莞市人民医院 | Monitoring and evaluating system and method for clinical application of flora drug resistance |
| CN115452804B (en) * | 2022-11-10 | 2023-01-31 | 泉州师范学院 | Raman spectrum detection device and method for body fluid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6379920B1 (en) * | 1999-07-24 | 2002-04-30 | Georgia Tech Research Corp. | Spectroscopic diagnostics for bacteria in biologic sample |
| CN105588827A (en) * | 2014-10-24 | 2016-05-18 | 中国科学院青岛生物能源与过程研究所 | Digital control system and digital control method for living single cell Raman analytic platform |
| CN107741417A (en) * | 2017-09-28 | 2018-02-27 | 上海合森生物科技有限公司 | A kind of method of quick detection cell biological processes in situ |
| CN107748158A (en) * | 2017-11-03 | 2018-03-02 | 中国科学院重庆绿色智能技术研究院 | A kind of microscopic Raman imaging spectral device for fast detecting and method |
-
2018
- 2018-09-27 CN CN201811129674.6A patent/CN109266717B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6379920B1 (en) * | 1999-07-24 | 2002-04-30 | Georgia Tech Research Corp. | Spectroscopic diagnostics for bacteria in biologic sample |
| CN105588827A (en) * | 2014-10-24 | 2016-05-18 | 中国科学院青岛生物能源与过程研究所 | Digital control system and digital control method for living single cell Raman analytic platform |
| CN107741417A (en) * | 2017-09-28 | 2018-02-27 | 上海合森生物科技有限公司 | A kind of method of quick detection cell biological processes in situ |
| CN107748158A (en) * | 2017-11-03 | 2018-03-02 | 中国科学院重庆绿色智能技术研究院 | A kind of microscopic Raman imaging spectral device for fast detecting and method |
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