CN105115970A - Phenotypic identification method and mass spectrometry method comprehensive microorganism identification system and phenotypic identification method and mass spectrometry method comprehensive microorganism identification method - Google Patents
Phenotypic identification method and mass spectrometry method comprehensive microorganism identification system and phenotypic identification method and mass spectrometry method comprehensive microorganism identification method Download PDFInfo
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Abstract
The invention relates to a phenotypic identification method and mass spectrometry method comprehensive microorganism identification system and a phenotypic identification method and mass spectrometry method comprehensive microorganism identification method. The system comprises: a matrix-assisted laser desorption ionization ion source for emitting laser to accelerate ions and lead out, and then carrying out ion focusing; a time-of-flight mass analyzer for making the ions pass through a field-free flight zone and detecting the electrical signals generated under the ion impact effect to form a mass spectrum; a microorganism image acquisition device for acquiring image information of a sample to be detected to obtain phenotypic characteristics; and a computer processing device connected with the devices, wherein the computer processing device comprises a control module, a signal acquisition module and an information processing module, and is used for carrying out machine vision-based pattern recognition according to the acquired image information to obtain the phenotypic identification result of the microorganism and carrying out specific-level and specific-range mass spectrometry identification according to the acquired the mass spectrum and phenotypic identification result combination to obtain the identification result. According to the present invention, the phenotypic identification method and the mass spectrometry method are organically combined so as to obtain the more efficient and accurate identification result compared with the identification result of the traditional method.
Description
Technical field
The present invention relates to field of microbial identification, particularly a kind of phenetics identification method and the comprehensive microbial identification system of mass spectroscopy and method.
Background technology
Machine vision (MachineVision) refers to capture target image by image-pickup device, image is sent to processor, based on pixel distribution and the information such as brightness, color, carry out the isoparametric differentiation of size, shape and color, and then draw differentiation result.China's machine vision applications originates from the eighties in 20th century, and development was swift and violent in recent years, and the market growth is remarkable, but machine vision technique is mainly used in the industrial circles such as electronic manufacture, automobile, pharmacy and package packing machine at present, occupies its market share of 70%.Pertinent literature has patent " a kind of automobile anti-collision method of machine vision and system ", master thesis " the electric whiteboard system key technology research based on machine vision " etc.At biological and medical field, machine vision technique starts new trial and exploration, and pertinent literature has: patent " a kind of Medical infusion velocity monitoring based on machine vision and control device ", master thesis " medical diagnosis on disease based on face coloured image is studied " etc.In field of microbial identification, machine vision pertinent literature is less, have patent " intelligent water treatment micro-organism machine vision identification system and method ", master thesis " research of food microorganisms rapid detection system ", master thesis " micro-image recognition technology detects the research of food bacteria total number fast " etc., but above application is mainly carried out quantity statistics based on machine vision technique to microorganism thus is carried out the quality analysis of target.
Mass spectroscopy has the features such as fast, highly sensitive, the qualitative ability of analysis speed is strong.In field of microbial identification, Matrix Assisted Laser Desorption/ionization time of flight mass spectrometry method (Matrix-assistedlaserdesorption/ionizationtime-of-flightm assspectrometry, MALDI-TOFMS) is a kind of important authentication method.This technology adds matrix in microorganism, laser ionization cell lysate (small protein or polypeptide) forms peptide mapping fingerprinting (Peptiedmassfingerprinting, PMF), compare with standard spectrum storehouse, thus realize the qualification of microorganism.Pertinent literature has patent " MALDI-TOF-MS detects the method for kinds of pathogenic vibrio ", master thesis " MALDIBiotyper microorganism Rapid identification and classification new technology " etc., not yet retrieves the correlation technique of machine vision phenetics authentication method and Mass Spectrometric Identification method connected applications.
Summary of the invention
Fundamental purpose of the present invention is to propose a kind of phenetics identification method and the comprehensive microbial identification system of mass spectroscopy and method, machine vision technique is adopted to carry out phenetics qualification according to the image information of microorganism, carry out the Molecular Identification of specific rank and scope according to peptide mass fingerprinting spectrum (PMF) that qualification result connexus spectrometry obtains, thus obtain more efficiently, microbial identification result more accurately.
The present invention adopts following technical scheme:
Phenetics identification method and the comprehensive microbial identification system of mass spectroscopy, is characterized in that: comprise
Matrix Assisted Laser Desorption ionization ion source, for the testing sample Emission Lasers scribbling matrix, makes it ionization and produces ion, accelerated by ion and draw, then carrying out ion focus;
Time of flight mass analyzer, for making ion by field-free flight district, and the electric signal produced under detecting ionic bombardment effect, form mass spectrogram;
Microorganism image acquiring device, for obtaining the image information of testing sample, obtains its phenotypic characteristic;
Computer processor unit, is connected with microorganism image acquiring device with Matrix Assisted Laser Desorption ionization ion source, time of flight mass analyzer, comprises control module, signal acquisition module and message processing module; This control module is for controlling the duty of Matrix Assisted Laser Desorption ionization ion source, time of flight mass analyzer and microorganism image acquiring device; This signal acquisition module is for gathering mass spectrogram and the image information of testing sample; The image information that this message processing module is used for according to gathering carries out the phenetics qualification result obtaining microorganism based on the pattern-recognition of machine vision, and carry out the mass spectroscopy qualification of specific rank, particular range in conjunction with phenetics qualification result according to the mass spectrogram gathered, finally obtain qualification result.
Preferably, described Matrix Assisted Laser Desorption ionization ion source includes: laser instrument, for Emission Lasers to irradiate the testing sample scribbling matrix, makes it ionization and produces ion; Accelerate high pressure and pulse extraction high pressure, for being carried out accelerating and drawing by ion; Ion focusing lenses, for carrying out ion focus.
Preferably, described time of flight mass analyzer comprises: field-free flight district, detecting device and data collecting card; This field-free flight district is used for for ion field-free flight, and this detecting device is used for producing electric signal under ionic bombardment, and this data collecting card is connected for gathering electric signal with detecting device.
Preferably, described microorganism image acquiring device comprises imageing sensor and image pick-up card, and this image pick-up card is connected for gathering image information with computer processor unit with imageing sensor.
Preferably, described imageing sensor adopts camera or camera.
Phenetics identification method and the comprehensive microorganism identification method of mass spectroscopy, is characterized in that: comprise the steps
1) obtain the image information of testing sample, carry out the phenetics qualification result obtaining microorganism based on the pattern-recognition of machine vision;
2) to the testing sample Emission Lasers scribbling matrix, make it ionization and produce ion, draw and carry out ion focus after ion is accelerated, and relief ion is by field-free flight district, and the electric signal produced under detecting ionic bombardment effect, form mass spectrogram;
3) obtain mass spectrogram and in conjunction with phenetics qualification result carry out specific rank, particular range mass spectroscopy qualification, finally obtain qualification result.
Preferably, in step 3) in, carry out classifying based on the pattern-recognition of machine vision according to the form of microorganism in image information and half-tone information.
Preferably, in step 2) in, described mass spectrogram is peptide mass fingerprinting spectrum.
Preferably, described microorganism comprises bacterium or viral or fungi.
From the above-mentioned description of this invention, compared with prior art, the present invention has following beneficial effect:
The present invention adopts and identify based on the phenetics of machine vision, performance microorganism conventional identification (phenetics qualification) simply, advantage fast, and do not rely on appraiser experience.Record microorganism phenotype picture, and carry out data with this and trace to the source.Mass spectrometer is utilized to gather microorganism peptide mapping fingerprinting (PMF), and the phenetics qualification result combined based on machine vision carries out specific rank, the mass spectroscopy qualification of particular range, accelerate the speed of qualification, make qualification more accurate, thus realize more efficiently and accurately identifying than current microbial identification system.
Accompanying drawing explanation
Fig. 1 is the composition schematic diagram of present system;
Fig. 2 is the schematic diagram of peptide fingerprinting of the present invention spectrum;
Fig. 3 is the schematic diagram that testing sample is positioned on target spot;
Wherein: 10, Matrix Assisted Laser Desorption ionization ion source, 11, laser instrument, 12, accelerate high pressure, 13, pulse draw high pressure, 14, ion focusing lenses, 20, time of flight mass analyzer, 21, field-free flight district, 22, detecting device, 23, data collecting card, 30, microorganism image acquiring device, 31, camera, 32, image pick-up card, 40, computer processor unit, 50, testing sample.
Embodiment
Below by way of embodiment, the invention will be further described.
With reference to Fig. 1, a kind of phenetics identification method and the comprehensive microbial identification system of mass spectroscopy, be provided with Matrix Assisted Laser Desorption ionization ion source (MALDI) 10, time of flight mass analyzer (TOF) 20, microorganism image acquiring device 30 and computer processor unit 40.This Matrix Assisted Laser Desorption ionization ion source (MALDI) 10 includes: laser instrument 11, for Emission Lasers to irradiate the testing sample 50 scribbling matrix, makes it ionization and produces ion; Accelerate high pressure 12 and pulse extraction high pressure 13, for being carried out accelerating and drawing by ion; Ion focusing lenses 14, for carrying out ion focus.
Time of flight mass analyzer (TOF) 20 includes field-free flight district 21, detecting device 22 and data collecting card 23, this field-free flight district 21 is for supplying ion field-free flight, this detecting device 22 for producing electric signal under ionic bombardment, and this data collecting card 23 is connected with detecting device 22 and forms mass spectrogram for gathering electric signal.This mass spectrogram is peptide mass fingerprinting spectrum (PMF), and the time that the ion due to different mass-to-charge ratio arrives detecting device 22 is different, so mass spectrogram embodies the molecular characterization of testing sample 50, thus can carry out constituent analysis.
Microorganism image acquiring device 30 comprises imageing sensor and image pick-up card 32, this image pick-up card 32 is connected for gathering image information with imageing sensor with computer processor unit 40, and this imageing sensor can be the high definition imageing sensors such as camera 31, mobile phone or camera.
This computer processor unit 40 can adopt industrial computer, it is connected with microorganism image acquiring device 30 with Matrix Assisted Laser Desorption ionization ion source 10, time of flight mass analyzer 20, comprises control module, signal acquisition module, message processing module and human-computer interaction interface.This control module is for controlling the duty of Matrix Assisted Laser Desorption ionization ion source 10, time of flight mass analyzer 20 and microorganism image acquiring device 30; This signal acquisition module is for gathering mass spectrogram and the image information of testing sample 50; This human-computer interaction interface is for realizing the information interaction of user and computer processor unit; The image information that this message processing module is used for according to gathering carries out the phenetics qualification result obtaining microorganism based on the pattern-recognition of machine vision, and carry out the mass spectroscopy qualification of specific rank, particular range in conjunction with phenetics qualification result according to the mass spectrogram gathered, finally obtain qualification result.
Based on above-mentioned system, the present invention also proposes a kind of phenetics identification method and the comprehensive microorganism identification method of mass spectroscopy, comprises the steps:
1) image information of testing sample 50 is obtained by microorganism image acquiring device 30, computer processor unit 40 carries out according to the forms such as the edge of microorganism in image information, concavo-convex, color and luster and half-tone information the phenetics qualification result obtaining microorganism based on the pattern-recognition of machine vision, and preserve microorganism phenotypic map picture and qualification result thereof, carry out data according to this and trace to the source.
2) adopt the laser instrument 11 of Matrix Assisted Laser Desorption ionization ion source (MALDI) 10 to scribbling testing sample 50 Emission Lasers of matrix (with reference to Fig. 3, testing sample 50 is positioned on target spot), make it ionization and produce ion, drawn after ion accelerates by high pressure 13 respectively by acceleration high pressure 12 and pulse and draw and carry out ion focus, then allow ion by field-free flight district 21 by time of flight mass analyzer (TOF) 20, and produce electric signal under adopting detecting device 22 and data collecting card 23 to detect ionic bombardment effect, form mass spectrogram.
3) last, computer processor unit 40 obtains mass spectrogram and carries out the mass spectroscopy qualification of specific rank, particular range in conjunction with phenetics qualification result, finally obtains qualification result.This specific rank refers to that the microorganism rank (be generally and belong to rank) that aforesaid phenetics identification method can be determined, this particular range refer to peptide mass fingerprinting spectrum (PMF) peak number range, the mass range produced.
The microorganism that the present invention can be used for identifying has bacterium, virus, fungi etc.Below in conjunction with instantiation, the present invention is elaborated: what this sentenced Escherichia coli (Escherichiacoli) and Shigella dysenteriae (Shigelladysen-teriae) is accredited as example.Escherichia coli belongs to Escherichia, is Pseudomonas normal in human intestine, not pathogenic under normal circumstances.Shigella dysenteriae belongs to Shigella, usually enters alimentary canal with sewage and food per os and makes people infect dysentery.Both more easily obscure in traditional authentication method, when particularly utilizing mass spectroscopy to differentiate, because both peptide mass fingerprintings compose (PMF) closely, are therefore difficult to both correctly to identify.First the present invention is carried out imaging by the nutrient culture media (this sentences Mai Kangkai is example) of microorganism image acquiring device 30 pairs of Escherichia coli and Shigella dysenteriae and is sent to computer processor unit 40, and computer processor unit 40 carries out the pattern recognition classifier based on machine vision according to the forms such as the edge of microorganism in image, concavo-convex, color and luster and half-tone information.Escherichia coli bacterium colony pinkiness or redness, slightly convex, circular on Mai Kangkai flat board herein, this embodies the feature in phenetics of Escherichia, but can not carry out the correct qualification to planting or plant following rank according to this.In like manner, Shigella dysenteriae bacterium colony on Mai Kangkai flat board is water white transparency, median size, embodies the feature of Shigella in phenetics, but can not carry out the correct qualification to planting or plant following rank successively.The present invention utilizes above-mentioned CF feature, carries out the pattern-recognition based on machine vision, obtains the genus grade classification of microorganism, and preserves microorganism image, carries out data according to this and traces to the source or manually judge again, increases the trackability and reliability differentiated.Then, utilize mass spectroscopy to carry out Matrix Assisted Laser Desorption ionization ion source (MALDI) 10 to above-mentioned two kinds of microorganisms to ionize, and gather respective peptide mass fingerprinting spectrum (PMF) by time of flight mass analyzer (TOF) 20, in conjunction with phenetics qualification result, respectively specific rank is carried out to the PMF of Escherichia coli and the PMF of Shigella dysenteriae, namely the standard spectrum library searching of rank (Escherichia and Shigella) is belonged to, and carry out particular range and PMF peak number range according to respective PMF, PMF mass range carries out standard spectrum search, because search has scope, object, so qualification speed improves greatly, finally realize the quick and precisely qualification of microorganism.The present invention effectively avoids mass spectroscopy and phenetics identification method shortcoming separately, has played respective advantage and has organically combined, having achieved and identify more fast and more accurately than conventional identification method.
Above are only the specific embodiment of the present invention, but design concept of the present invention is not limited thereto, all changes utilizing this design the present invention to be carried out to unsubstantiality, all should belong to the behavior of invading scope.
Claims (9)
1. phenetics identification method and the comprehensive microbial identification system of mass spectroscopy, is characterized in that: comprise
Matrix Assisted Laser Desorption ionization ion source, for the testing sample Emission Lasers scribbling matrix, makes it ionization and produces ion, accelerated by ion and draw, then carrying out ion focus;
Time of flight mass analyzer, for making ion by field-free flight district, and the electric signal produced under detecting ionic bombardment effect, form mass spectrogram;
Microorganism image acquiring device, for obtaining the image information of testing sample, obtains its phenotypic characteristic;
Computer processor unit, is connected with microorganism image acquiring device with Matrix Assisted Laser Desorption ionization ion source, time of flight mass analyzer, comprises control module, signal acquisition module and message processing module; This control module is for controlling the duty of Matrix Assisted Laser Desorption ionization ion source, time of flight mass analyzer and microorganism image acquiring device; This signal acquisition module is for gathering mass spectrogram and the image information of testing sample; The image information that this message processing module is used for according to gathering carries out the phenetics qualification result obtaining microorganism based on the pattern-recognition of machine vision, and carry out the mass spectroscopy qualification of specific rank, particular range in conjunction with phenetics qualification result according to the mass spectrogram gathered, finally obtain qualification result.
2. a kind of phenetics identification method as claimed in claim 1 and the comprehensive microbial identification system of mass spectroscopy, it is characterized in that: described Matrix Assisted Laser Desorption ionization ion source includes: laser instrument, for Emission Lasers to irradiate the testing sample scribbling matrix, make it ionization and produce ion; Accelerate high pressure and pulse extraction high pressure, for being carried out accelerating and drawing by ion; Ion focusing lenses, for carrying out ion focus.
3. a kind of phenetics identification method as claimed in claim 1 and the comprehensive microbial identification system of mass spectroscopy, is characterized in that: described time of flight mass analyzer comprises: field-free flight district, detecting device and data collecting card; This field-free flight district is used for for ion field-free flight, and this detecting device is used for producing electric signal under ionic bombardment, and this data collecting card is connected for gathering electric signal with detecting device.
4. a kind of phenetics identification method as claimed in claim 1 and the comprehensive microbial identification system of mass spectroscopy, it is characterized in that: described microorganism image acquiring device comprises imageing sensor and image pick-up card, and this image pick-up card is connected for gathering image information with computer processor unit with imageing sensor.
5. a kind of phenetics identification method as claimed in claim 4 and the comprehensive microbial identification system of mass spectroscopy, is characterized in that: described imageing sensor adopts camera or camera.
6. phenetics identification method and the comprehensive microorganism identification method of mass spectroscopy, is characterized in that: comprise the steps
1) obtain the image information of testing sample, carry out the phenetics qualification result obtaining microorganism based on the pattern-recognition of machine vision;
2) to the testing sample Emission Lasers scribbling matrix, make it ionization and produce ion, draw and carry out ion focus after ion is accelerated, and relief ion is by field-free flight district, and the electric signal produced under detecting ionic bombardment effect, form mass spectrogram;
3) obtain mass spectrogram and in conjunction with phenetics qualification result carry out specific rank, particular range mass spectroscopy qualification, finally obtain qualification result.
7. a kind of phenetics identification method as claimed in claim 6 and the comprehensive microorganism identification method of mass spectroscopy, it is characterized in that: in step 3) in, carry out classifying based on the pattern-recognition of machine vision according to the form of microorganism in image information and half-tone information.
8. a kind of phenetics identification method as claimed in claim 6 and the comprehensive microorganism identification method of mass spectroscopy, is characterized in that: in step 2) in, described mass spectrogram is peptide mass fingerprinting spectrum.
9. a kind of phenetics identification method as claimed in claim 6 and the comprehensive microorganism identification method of mass spectroscopy, is characterized in that: described microorganism comprises bacterium or virus or fungi.
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| CN111344572A (en) * | 2017-11-14 | 2020-06-26 | 株式会社诺斯凯斯特 | Antibiotic resistance distinguishing device and method based on MALDI-TOF mass analysis |
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