CN109765287A - A kind of microorganism rapid identification method of cell ejection sorting and mass spectrometry - Google Patents
A kind of microorganism rapid identification method of cell ejection sorting and mass spectrometry Download PDFInfo
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- CN109765287A CN109765287A CN201910089027.5A CN201910089027A CN109765287A CN 109765287 A CN109765287 A CN 109765287A CN 201910089027 A CN201910089027 A CN 201910089027A CN 109765287 A CN109765287 A CN 109765287A
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Abstract
The present invention provides a kind of cell ejection sorting and the microorganism rapid identification method of mass spectrometry, include the following steps: known to obtaining include or may include microorganism sample to be tested；By sample to be tested point sample in the sample chip of unicellular ejection sorter；Microorganism in sample chip is identified, and will be identified as in the microorganism ejection sorting to mass spectrometric target plate of certain amount of same kind；The microorganism kind sub-elected by mass spectrography identification ejection.The present invention substantially reduces mass spectrum sample and prepares the consumed time, greatly accelerates the identification speed of microorganism, and also can be carried out to the microorganism for being difficult to cultivate and fast and accurately identify.
Description
Technical field
The present invention relates to a kind of microorganism identification method, especially a kind of microorganism of cell ejection sorting and mass spectrometry
Rapid identification method.
Background technique
Fast and accurately microbial identification is in clinical diagnosis, environmental sanitation monitoring, Food Safety Analysis, biological industry mistake
It is of great significance in journey detection and biological medicine research.Especially in the diagnosis of clinical infectious disease, micro- life is infected
Quick, the errorless identification of object has directive function for doctor's clinical application, closely bound up with patient vitals and prognosis.
Traditional is very long with the microbial identification time-consuming of biochemical analysis method based on cultivating, it usually needs several days time, pole has
Patient's treatment may be therefore delayed.Matrix-assisted laser desorption ionization (MALDI-TOF MS) is applied in recent years
In the strain idenfication of clinical pathogenic microorganism, strain mirror is carried out according to the characteristic protein finger-print of different microorganisms
It is fixed.MALDI-TOF MS analytic approach is a kind of analysis side by being analyzed the measurement of sample ion mass-to-charge ratio
Method, the principle is as follows: sample and matrix mixing point being formed cocrystallization on metallic plates, use a laser as energy source spoke
Crystalline solid is penetrated, substrate molecule, which absorbs energy, to be adsorbed sample and make its ionization, and the charged ion of different mass-to-charge ratioes is generated；Sample from
Son obtains identical kinetic energy under accelerating field, carries out quality point into flying time mass spectrum analysis device after accelerating through high pressure, focusing
Analysis；The mass-to-charge ratio (m/z) of ion and flight time it is square directly proportional, handled through computer, quality map be depicted as, by soft
Part analysis is compared, and screens and determine specific manner, to realize the differentiation and identification to objective microbe kind or bacterial strain.Example
Such as, a kind of method that the patent document of Publication No. CN103616434A discloses Mass Spectrometric Identification microorganism, to microorganism
Identification accuracy rate reach 99% or more, effectively reduce the probability of erroneous judgement and wrong report, also reduce the mistake due to microorganism
Sentence and report by mistake bring safety accident.
Although the period of mass spectrography pathogen identification largely shortens compared with conventional biochemical analytic approach,
Since mass spectrography is more demanding to microbiological purity, for the test sample that clinical sample etc. includes multiple-microorganism, still
It needs to take a long time and turns out pure bacterium colony from sample to be tested, this incubation usually requires 18-24 hours, for life
Long slow microorganism even needs the longer time.The patent document of Publication No. CN109060743A discloses a kind of micro- life
Object identification method forms characteristic spectrum using the fluorescence intensity change generated between functionalization carbon dots and microorganism, and passes through
Mathematical statistics method realizes the identification to microorganism, although it does not need instrument costly, the method is to microorganism
Identifying type and accuracy has considerable restraint, and it also needs to cultivate microorganism when implementing, and identifies time shortening
It is limited.The patent document of Publication No. CN104136908A also discloses a kind of for identifying the spectrum hand of microorganism in culture
Section and method, also need to carry out the biological sample of acquisition in blood agar plate culture in 12 to 24 hours, this is resulted in
The whole extension for identifying the time of microorganism.
In addition, importantly, just can not for the microorganisms for being difficult to grow in the medium certain in sample to be tested
It is identified using the methods of mass spectrum, this has seriously affected mass-spectrometric technique to the speed and accuracy rate of detection of pathogens.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of microorganisms of cell ejection sorting and mass spectrometry
Rapid identification method substantially reduces mass spectrum sample and prepares the consumed time, greatly accelerates the identification speed of microorganism, and
The microorganism for being difficult to cultivate also can be carried out and fast and accurately identify.
The technical solution adopted by the present invention to solve the technical problems is: a kind of cell ejection sorting is micro- with mass spectrometry
Biological rapid identification method, includes the following steps:
Step a: including known to acquisition or may include the sample to be tested of microorganism；
Step b: by sample to be tested point sample in the sample chip of unicellular ejection sorter；
Step c: identifying the microorganism in sample chip, and will be identified as the microorganism of the certain amount of same kind
In ejection sorting to mass spectrometric target plate；
Step d: the microorganism kind sub-elected by mass spectrography identification ejection.
Embodiment as a further preference, the step b further include operating as follows: by microorganism from described to test sample
It separates and/or is enriched in product；
Embodiment as a further preference, the sample to be tested are selected from blood, blood plasma, serum, urine, saliva, group
Knit liquid, joint fluid, sperm, cerebrospinal fluid, bone marrow aspiration liquid, gastric content, peritoneal lavage fluid, phlegm, bronchus purulent secretion, group
Knit homogenate, bone homogenate, vaginal fluid, aspirate, swab and swab slag liquid.
Embodiment as a further preference, the sample to be tested are selected from food, drug, pharmaceutical raw material, cosmetics, drink
With water, undrinkable water, soil and vegetable material.
Embodiment as a further preference, it is described that microorganism separated and/or be enriched with from the sample to be tested
Method includes but is not limited to: filtration method, centrifugal process, separation by precipitation, Magnet Treatment, paramagnetic particle method, electric field separates method, immune richness
Collection method, cultivation.
Embodiment as a further preference, the microorganism in sample chip carry out know method for distinguishing include but
It is not limited to: Raman spectroscopy, fluorescence signal detection, morphological analysis method, micro-imaging method, biochemical analysis method, immunoassay
Method, nucleic acid probe method.
Embodiment as a further preference, the step d are specifically included: matrix liquid is added；It is sub-elected described in acquisition
Microorganism mass spectrogram；By being compared with standard reference mass spectrogram, the kind information of detected microorganism is obtained.
The positive effect of the present invention: the present invention obtains pure micropopulation using cell ejection method for separating, is substituted in this
Time-consuming longest microculture step in Mass Spectrometer Method so that mass spectrography qualification time foreshortened to from least one day 12 hours it
It is interior, the integral cycle for being delivered to from sample to be tested and providing qualification result report is greatly shortened, and the present invention is to being difficult to train
Feeding microorganism, which also can be carried out, fast and accurately identifies, is of great significance to analysis of clinic pathogenic microorganism diagnosis, while the present invention also fits
For the fields such as level of pollution, quality control in animal doctor's detection and real-time monitoring industrial environment.
Specific embodiment
Below to a preferred embodiment of the present invention will be described in detail.
The present invention provides the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry, including walks as follows
It is rapid:
Step 1: include known to acquisition or may include microorganism sample to be tested；
Step 2: microorganism being separated from the sample to be tested, and the microorganism isolated is enriched with；
Step 3: by the microorganism point sample of enrichment in the sample chip of unicellular ejection sorter；
Step 4: same category identification is carried out to the microorganism in sample chip, and the certain amount of same kind will be identified as
In microorganism ejection sorting to mass spectrometric target plate；
Step 5: the microorganism kind sub-elected by mass spectrography identification ejection.
Wherein, the operation for microorganism being separated and/or being enriched in step 2 refers to by implementing separation and/or enrichment
Step separates the microorganism in test sample with the other components (such as non-microorganism or its component) in sample, and by micro- life
Object enrichment is identification and density needed for characterization.Wherein, separation can be not up to 100%, as long as microorganism proportion is not done
Disturb microorganism detection.The method for separating and concentrating includes point of all microorganisms suitable for various forms sample to be tested
From and/or enrichment method, including but not limited to centrifugal process, filtration method etc..In addition, for can inherently reach identification require to
Sample can also be directly used in subsequent authentication step without isolation and/or enriching step (i.e. step 2 can be omitted).
The method (whether characterization microorganism is same kind of method) with category identification is carried out to microorganism in step 4,
Raman spectroscopy, fluorescence signal detection, morphological analysis including but not limited in existing method etc., preferably Raman spectroscopy.
Ejection sorting (unicellular ejection sort) described in step 4, refer to by certain amount be identified as it is same kind of
Microbic shot is incident upon on mass spectrometric target plate, in region of the specific ejectable to target plate center for a specified size of origin,
Such as a diameter is within the scope of the border circular areas of 0.01mm-10mm.
The sample to be tested can be for presence or there may be the clinical samples of microorganism, non-clinical sample, and to micro-
The presence of biology and/or kind carry out material sample that is conventional or irregularly detecting.
The clinical sample can be any type sample usually tested in clinical or research laboratory, including but not
It is limited to blood, blood plasma, serum, blood part, urine, saliva, tissue fluid, joint fluid, sperm, cerebrospinal fluid, bone marrow aspiration liquid, stomach
Content, peritoneal lavage fluid, phlegm, bronchus purulent secretion, tissue homogenate, bone homogenate, vaginal fluid, aspirate, swab
And swab slag liquid, other body fluid etc..
The non-clinical sample can be various substances, including but not limited to Edible material, food (including drink), medicine
Object, pharmaceutical raw material, cosmetics, drinking water, undrinkable water, waste water, sewage, environmental water sample are (for example, seawater, river water, lake water, well
Water etc.), air, soil, vegetable material (for example, root, stem, leaf, flower, fruit, seed etc.), blood product (such as serum, blood
Slurry, blood platelet, white blood cell fraction etc.), organ donation or tissue sample, biological warfare sample etc..
Embodiment 1
The preferred embodiment of the present invention 1 provides the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry,
Include the following steps:
Step 1: include known to acquisition or may include microorganism sample to be tested；
Step 2: by sample to be tested point sample in the sample chip of unicellular ejection sorter；
Step 3: same Species identification being carried out to the microorganism in sample chip using Raman spectroscopy, specific steps include:
1) the Raman map of microbial single-cell is obtained；
2) clustering is carried out using respective algorithms；
3) the single celled position of quasi-microorganism of the same race is marked, is prepared for cell ejection sorting.
Step 4: will be identified as in the microorganism ejection sorting to mass spectrometric target plate of the certain amount of same kind, specifically may be used
Ejection is to using the center of metallic plates as in the border circular areas of origin；
Step 5: the microorganism kind sub-elected by mass spectrography identification ejection, concrete operations include:
1) a certain amount of matrix liquid is added in the central area Xiang Suoshu；
2) mass spectrogram of tested microorganism is obtained；
3) by being compared with standard reference mass spectrogram, the kind information of detected microorganism is obtained.
Embodiment 2
The preferred embodiment of the present invention 2 provides the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry,
Include the following steps:
Step 1: include known to acquisition or may include microorganism sample to be tested；
Step 2: microorganism being separated from the sample to be tested, and the microorganism isolated is subjected to richness using centrifugal process
Collection；
Step 3: by the microorganism point sample of enrichment in the sample chip of unicellular ejection sorter；
Step 4: same to Species identification, specific steps packet being carried out to the microorganism in sample chip using fluorescence signal detection
It includes:
1) it is marked before sample chip using microorganism of the fluorescent dye to enrichment in the microorganism point sample that will be enriched with
Note, fluorescent dye and can have to be all with components a certain or certain in microorganism specificity or non-specific binding
And/or generate fluorescence dyestuff, including but not limited to Nile Red, SYPRO, Pyronin Y, SYBR Green, Hoechst,
DAPI, RedoxSensor, PI, 2-NBDG, CFDA, DiBac4, Ethidium Bromide, GFP, YFP or autofluorescence albumen
Deng；
2) by the microbiological specimens point sample after the microbiological specimens or fluorescent marker of included fluorescin in sample chip；
3) it is positioned by image recognition to unicellular；
4) according to the property of autofluorescence albumen or fluorescent dye, select suitable excitation wavelength and launch wavelength detect to
The fluorescence signal of micrometer biology, selectable excitation wavelength include but is not limited to 350-400nm, 460-500nm, 520-580nm,
Selectable launch wavelength includes but is not limited to 400-500nm, 500-560nm, 580-650nm；
5) clustering is carried out using respective algorithms；
6) the single celled position of quasi-microorganism of the same race is marked, is prepared for cell ejection sorting.
Step 5: will be identified as in the microorganism ejection sorting to mass spectrometric target plate of the certain amount of same kind, specifically may be used
Ejection is to using the center of metallic plates as in the border circular areas of origin；
Step 6: the microorganism kind sub-elected by mass spectrography identification ejection, concrete operations include:
1) a certain amount of matrix liquid is added in the central area Xiang Suoshu；
2) mass spectrogram of tested microorganism is obtained；
3) by being compared with standard reference mass spectrogram, the kind information of detected microorganism is obtained.
Embodiment 3
The preferred embodiment of the present invention 3 provides the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry,
Include the following steps:
Step 1: include known to acquisition or may include microorganism sample to be tested；
Step 2: microorganism being separated from the sample to be tested, and the microorganism isolated is subjected to richness using filtration method
Collection；
Step 3: by the microorganism point sample of enrichment in the sample chip of unicellular ejection sorter；
Step 4: same to category identification, specific steps packet being carried out to the microorganism in sample chip using morphological analysis method
It includes:
1) optional step: in the microorganism point sample that will be enriched with before sample chip, using dyestuff to microorganism to be detected
It is dyed, optional dyestuff includes but is not limited to: methylene blue, crystal violet, crystal violet, basic fuchsin, dimethyl diaminophenazine chloride, peacock green, luxuriant
Red, Yihong, Congo red, algae red, nigrosine, picric acid, acid fuchsin, auspicious Tuo Shi (Wright) dyestuff, Ji Musashi (Gimsa)
Dyestuff, purple red class dyestuff etc.；
2) the microbiological specimens point sample to be detected after being unstained or dye is in sample chip；
3) by image recognition algorithm, classification analysis is carried out to microbial single-cell form according to parameters such as character, colors；
4) the single celled position of microorganism belonging to genus of the same race is marked, is prepared for cell ejection sorting.
Step 5: will be identified as in the microorganism ejection sorting to mass spectrometric target plate of congener certain amount, specifically may be used
Ejection is to using the center of metallic plates as in the region of origin；
Step 6: the microorganism kind sub-elected by mass spectrography identification ejection, concrete operations include:
1) a certain amount of matrix liquid is added in the central area Xiang Suoshu；
2) mass spectrogram of tested microorganism is obtained；
3) by being compared with standard reference mass spectrogram, the kind information of detected microorganism is obtained.
Of the invention innovative is combined cell ejection sorting with mass spectrum, can be used for quickly reflecting to microorganism
Not.Compared with prior art, the cracking cell ejection method for separating of operating speed of the present invention obtains pure micropopulation, is replaced with this
Time-consuming longest microculture step, provides identification to greatly shorten and be delivered to from sample to be tested in Mass Spectrometer Method
As a result it can be foreshortened within 12 hours from current at least 1 day the whole qualification cycle reported.In addition, in test sample
Due to being difficult to cultivate and can not carry out the microorganism of Mass Spectrometer Method, the present invention also can quick and precisely identify it.
Application field of the invention includes but is not limited to clinical diagnosis, animal doctor's detection, industrial pollution detection, food quality control
The fields such as system.
It is above-described to be merely a preferred embodiment of the present invention, it should be understood that the explanation of above embodiments is only used
In facilitating the understanding of the method and its core concept of the invention, it is not intended to limit the scope of protection of the present invention, it is all of the invention
Any modification for being made within thought and principle, equivalent replacement etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of microorganism rapid identification method of cell ejection sorting and mass spectrometry, which comprises the steps of:
Step a: including known to acquisition or may include the sample to be tested of microorganism；
Step b: by sample to be tested point sample in the sample chip of unicellular ejection sorter；
Step c: identifying the microorganism in sample chip, and will be determined as the microorganism ejection of the certain amount of same kind
On sorting to mass spectrometric target plate；
Step d: the microorganism kind sub-elected by mass spectrography identification ejection.
2. the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry according to claim 1, special
Sign is that the step b further includes operating as follows: microorganism is separated and/or is enriched with from the sample to be tested.
3. the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry according to claim 1, special
Sign is: the sample to be tested is selected from blood, blood plasma, serum, urine, saliva, tissue fluid, joint fluid, sperm, cerebrospinal fluid, bone
Marrow extract, gastric content, peritoneal lavage fluid, phlegm, bronchus purulent secretion, tissue homogenate, bone homogenate, vaginal fluid,
One or more of aspirate, swab and swab slag liquid.
4. the microorganism rapid identification method of a kind of cell ejection sorting and mass spectrometry according to claim 1, special
Sign is: the sample to be tested is selected from food, drug, pharmaceutical raw material, cosmetics, drinking water, undrinkable water, soil and plant material
One or more of material.
5. the microorganism Rapid identification side of a kind of cell ejection sorting and mass spectrometry according to claim 1 or 2 or 3
Method, it is characterised in that: the method that the microorganism by sample to be tested is separated and/or is enriched be filtration method, centrifugal process,
One of separation by precipitation, Magnet Treatment, paramagnetic particle method, electric field separates method, immunity enrichment method, cultivation or combination.
6. the microorganism Rapid identification side of a kind of cell ejection sorting and mass spectrometry according to claim 1 or 2 or 3
Method, it is characterised in that: the microorganism in sample chip carries out being Raman spectroscopy, fluorescence letter with the method for category identification
One of number detection method, morphological analysis method, micro-imaging method, biochemical analysis method, immunoassay, nucleic acid probe method or group
It closes.
7. the microorganism Rapid identification side of a kind of cell ejection sorting and mass spectrometry according to claim 1 or 2 or 3
Method, it is characterised in that: the step d is specifically included: matrix liquid is added；The mass spectrogram of the microorganism sub-elected described in acquisition；It is logical
It crosses and is compared with standard reference mass spectrogram, obtain the kind information of detected microorganism.
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| CN110887892A (en) * | 2019-12-23 | 2020-03-17 | 复旦大学 | Mass spectrum detection method for small amount of samples |
| CN112080430A (en) * | 2020-09-22 | 2020-12-15 | 长春长光辰英生物科学仪器有限公司 | Method for processing cell sample in single cell sorting process |
| CN113984659A (en) * | 2021-10-13 | 2022-01-28 | 自然资源部第二海洋研究所 | Aerobic non-oxygen-producing photosynthetic bacterium detection method based on single-cell Raman spectrum |
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| CN103353452A (en) * | 2013-07-12 | 2013-10-16 | 北京惟馨雨生物科技有限公司 | Cell carrier chip and single cell rapid identifying or sorting method employing same |
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Application publication date: 20190517 |