CN107024530B - Method that detection microorganism is composed by internal standard material and products thereof - Google Patents
Method that detection microorganism is composed by internal standard material and products thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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Abstract
The present invention provide it is a kind of by internal standard object come the method for Mass Spectrometer Method microorganism, which has known molecular weight and mass-to-charge ratio, and the peak value spectrogram of microorganism characteristic protein is not disturbed during microorganism detection.The present invention also provides internal standard object combination, reagent composition and its coherent detection products.The method of internal standard material correction single microbial sample detection is utilized in the present invention, it is applicable to include fungi, in all kinds of microbiological specimens for MALDI TOF MS detections such as bacterium, the accuracy rate of microbial identification is improved, while compensates for and only has external perimysium reference object to do the deficiency corrected currently on the market.
Description
Technical field
The invention belongs to biological technical fields, are related in the detection of flight time mass spectrum system detectio microbiological specimens
Calibration is just.
Background technology
Matrix-Assisted Laser Desorption Ionization Time of Flight (Matrix-assisted laser desorption
Lionization time of flight mass spectrometry, MALDI-TOF-MS) technology is rapidly to send out in recent years
One of the proteomics of exhibition, genomics technologies have the characteristics that high sensitivity, accuracy be high and high resolution, are life
The scientific research analysis means of testing quick, high-throughput with the clinical offers such as major disease early warning and auxiliary diagnosis be also simultaneously
A kind of method of good identification pathogenic microorganism.
The principle of MALDI-TOF-MS is to use laser irradiating sample and substrate formed cocrystallization film, and matrix is from laser
Energy transmission is absorbed to biomolecule, and obtains proton by proton translocation to biomolecule or from biomolecule in ionization process,
And the process for ionizing biomolecule.The principle of TOF is that ion accelerates to fly over dirft tube under electric field action, is examined according to reaching
The flight time for surveying device it is different and be detected measure ion mass-to-charge ratio (M/Z) it is directly proportional to the flight time of ion, detect
Ion.Although the accuracy of MALDI-TOF-MS is up to 0.1%~0.01%, the significantly larger than current SDS electrophoresis routinely applied
With high productivity computing technology.MALDI-TOF MS technologies have the characteristics that quick, accurate, stable, high-throughput, low cost；
MALDI-TOF MS technologies are the quick analytical technology of routine of Clinical microorganism and one of the analytical technology of drug-resistant microorganism；
MALDI-TOF MS technologies provide effective identification technology for the structure of pathogen resources bank and support.But due to systematic error
Etc. factors influence the accurate quantification of experimental result, therefore in Mass Spectrometer Method sample, introduce the internal standard of known concentration and ratio, from
And improve accuracy and the precision of single microbial sample identification.
The method of curve matching at home and abroad has more universal application, Jiangyue Wu etc. at present
(Anal.Chem.1997,69,3767-3771) cyclosporin is quantified using the matched curve method of MALDI-TOF-MS
Test；Mazarin etc. (Anal.Chem.2006,78,2758-2764) combines pulsed gradient spinning NMR and MALDI-
When TOF-MS quantitative determines polymer, equally employ matched curve and carry out Data correction；2009, Lv Shuan et al. utilized fitting
Curve successfully develops a kind of MALDI-TOF-MS quantitative approach of Phosphorylated Peptide.
The technology is that the cracking of acidic matrix auxiliary cell, laser excitation cell lysate are added in complete microorganism
(little albumen or polypeptide) forms peptide mapping fingerprinting, and the general character reference spectrum storehouse of the genus and species level with having built is compared,
So as to fulfill the identification of microorganism.And the testing result of various features peptide or peptide fingerprint has specific mass-to-charge ratio by a series of
(M/Z) peak curve is identified.It is protein fingerprint spectrum in the accuracy using MALDI-TOF MS mass spectrograph collecting samples
Microorganism identifies the key factor of system, if the impurity or systematic error due to cell lysate in detection process (continue
Supplement) influence, the peak curve of testing result can be caused a degree of error (such as the peak of 1-10 Da molecular weight occur
Value offset), therefore generally require and be corrected by reference substance, so that measuring peak curve and true peak curves.
Yuan Xianglin etc. (《Analytical chemistry research report》, the 1st phase of volume 29 in 2001) and it reports in MATRIX-ASSISTED LASER solution
Ionization time of flight mass spectrometry is inhaled in the quantitative analysis of Ginsenoside Rg3, rutin being selected as internal standard compound, in reappearance and line
Property aspect be better than gossypose.The raising of resolution ratio and testing concentration is represented with relative peak area, can make it is averagely opposite by mistake
Difference is substantially reduced, and improves quantitative result.However, this method is not intended in Mass Spectrometer Method microorganism, examined with MALDI-TOF MS
It is reflective-mode used by the quantitative analysis of Ginsenoside Rg3 unlike the linear model that micrometer biology uses, and it is to be measured
The detection range of sample is less than 1000Da, while its detection interval is only limitted to 400-900 (M/Z), can not meet the inspection of microorganism
Section is surveyed, therefore is restricted.
Chinese patent application 2014100902157, denomination of invention are public " for the polypeptide reference substance of early diabetes diagnosis "
A kind of internal standard polypeptide for Mass Spectrometer Method early diabetes is opened, which derives from human serum albumins, has 19 ammonia
Base acid sequence.It needs by cycle longer incubation time, while the internal standard peptide fragment judges to participate in result, by calculating mesh
The ratio of peptide fragment and internal standard peptide fragment is marked to judge the testing result of the risk possibility rather than judgement sample of having diabetes in itself
Whether detect accurate.However, this method is equally not intended in MALDI-TOF MS detection microorganisms, detection range is respectively less than
1000Da differs more with microorganism detection scope 2000Da-20000Da, can not meet the detection interval of microorganism, therefore by
Limitation is arrived.
As the immediate prior art, " microbial identification is used for Chinese patent application 201510246677.8, denomination of invention
Mass spectrograph molecular weight calibration standard items and preparation method and application " disclose one group using mass-to-charge ratio m/z be respectively 2094,
2466th, 3149,4364 etc. 18 Escherichia coli characteristic proteins for correction feature spectrogram, stablize correction effect as reference substance
Fruit.However, although the reference substance involved in the invention can effectively detect microorganism, it must be carried out before Mass Spectrometer Method
Parallel testing, to carry out molecular weight calibration to mass spectrograph and can not directly judge the accuracy of single sample testing result, still
So belong to external perimysium reference object, step is added in detection process, be unfavorable for the high-throughput, quick, just of a large amount of micro-biological samples
Prompt detection.In conclusion to single when the mass spectrography detection sample standard deviation reported at present cannot meet identification micro-biological samples
The correction of pattern detection, wherein MALDI-TOF MS methods only in for quantitative determination using addition in calibration method, and for
MALDI-TOF MS methods identification microorganism there is no report.
At present in the market use MALDI-TOF-MS methods detection microorganism during, detection range is mainly
2000-20000Da (Calderaro etc.), although having been reported that (Pignone etc., Shah etc., Kumar etc., Edwards-Jones
Deng) mass spectra peak of 400-3000Da is used for the research for differentiating parting etc., but the characteristic peak in the mass range not by with
In the identification of commercially available MALDI-TOF-MS instruments.And in 2000-20000Da detection ranges, the principal character peak of microorganism is small
In 12000Da (Winkler etc.), therefore in the research of existing laser mass spectrometry detection microorganism, still using external perimysium reference
Object, even a variety of external perimysium reference objects are corrected, so as to add detection time and testing cost.
Therefore, a kind of new method by flight time mass spectrum system detectio microorganism characteristic protein is needed at present.
The content of the invention
The principle of the invention is：Mass-to-charge ratio section (the 3000- of microorganism is detected according to existing MALDI-TOF-MS
13000m/z), it is put forward for the first time using less than 3000m/z or the single internal standard object more than 12000m/z, the internal standard object
With known molecular weight and mass-to-charge ratio, the peak value of microorganism characteristic protein is not disturbed to compose during microorganism detection
Figure.Therefore, the internal standard object is with the addition of in each microbiological specimens to be checked, it is made to be produced simultaneously with each microbiological specimens
Raw specific mass spectrogram, and pass through the known molecular amount of the reference substance and its corresponding characteristic peak, to correct each microorganism sample
The mass spectrogram of product (is tested microorganism characteristic protein for example, according to the testing molecule amount of reference substance and the difference of actual molecular weight
Molecule measuring magnitude be corrected, such as actual measured value is corrected according to the difference), so as to improve single microbial
The accuracy of sample identification and precision.Since the internal standard material for correction that the present invention selects is selected in 1000-
3000Da or 13000-20000Da can not only reach detection result, also on the basis of microbial identification is not influenced, add
The internal standard material added can correct the entire spectrogram of single sample, may be caused in the detection with making up MALDI-TOF MS
The deficiency of deviation.
Therefore, first purpose of the invention is to provide one kind by internal standard object come flight time mass spectrum detection microorganism
Method, this method includes：
(1) pre-treatment is carried out to microbiological specimens；
(2) 1000Da is added in the microbiological specimens of preceding processing<Average molecular weight<3000Da or 12000Da<Average mark
Son amount<The internal standard object of 20000Da；
(3) Mass Spectrometer Method will be carried out containing the microbiological specimens of internal standard object so that microbiological specimens and internal standard
Object generates specific mass spectrogram simultaneously, and passes through the known molecular amount of the reference substance and its corresponding characteristic peak, each to correct
The mass spectrogram of microbiological specimens, so as to obtain accurate testing result；
Wherein, the internal standard object can be in above-mentioned average molecular weight known polypeptide or/and protein standard substance or
It is combined.
In one embodiment, the internal standard object is selected from 1000Da<Average molecular weight<2000Da's is known more
Peptide or/and protein standard substance or its combination.In a specific embodiment, the internal standard object is selected from polypeptide P (33507-
63-0), molecular weight 1347.63Da, sequence are SEQ ID NO:1：RPKPQQFFGLM-NH2；Or, selected from polypeptide P14R
(synthetic peptide), molecular weight 1533.85Da, sequence are SEQ ID NO:2：PPPPPPPPPPPPPPR.
In one embodiment, the internal standard object is selected from 2000Da<Average molecular weight<3000Da's is known more
Peptide or/and protein standard substance or its combination.In a specific embodiment, the internal standard object is selected from polypeptide A CTH
Fragment18-39 (human), molecular weight 2465.19Da, sequence are SEQ ID NO:3：
RPVKVYPNGAEDESAEAFPLEF。
In one embodiment, the internal standard object is selected from 12000Da<Average molecular weight<180000Da's is known
Polypeptide or/and protein standard substance or its combination.In a specific embodiment, the internal standard object is selected from horse flesh red eggs
White Apomyoglobin (equine), molecular weight 16952Da, sequence are SEQ ID NO:4：
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILK
KKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG。
Also in other embodiments, the internal standard object is selected from any combination in above-mentioned average molecular weight range.
In one specific embodiment, wherein the internal standard object is selected from peptide material P (33507-63-0) and h-Mb
The combination of Apomyoglobin (equine).In another embodiment, the internal standard object is selected from P14R
The combination of (synthetic peptide) and h-Mb Apomyoglobin (equine)；In other specific embodiment parties
In case, the internal standard object is selected from ACTH fragment18-39 (human) and h-Mb Apomyoglobin
(equine) combination；And in other specific embodiment, the internal standard object is selected from selected from peptide material P
The combination of (33507-63-0) and ACTH fragment18-39 (human).
Second purpose of the invention is to provide a kind of internal standard object in the above method.
In one embodiment, the internal standard object is selected from 1000Da<Average molecular weight<2000Da's is known more
Peptide or/and protein standard substance or its combination.In a specific embodiment, the internal standard object is selected from polypeptide P (33507-
63-0), molecular weight 1347.63Da, sequence are SEQ ID NO:1：RPKPQQFFGLM-NH2；Or, selected from polypeptide P14R
(synthetic peptide), molecular weight 1533.85Da, sequence are SEQ ID NO:2：PPPPPPPPPPPPPPR.
In one embodiment, the internal standard object is selected from 2000Da<Average molecular weight<3000Da's is known more
Peptide or/and protein standard substance or its combination.In a specific embodiment, the internal standard object is selected from polypeptide A CTH
Fragment18-39 (human), molecular weight 2465.19Da, sequence are SEQ ID NO:3：
RPVKVYPNGAEDESAEAFPLEF。
In one embodiment, the internal standard object is selected from 12000Da<Average molecular weight<180000Da's is known
Polypeptide or/and protein standard substance or its combination.In a specific embodiment, the internal standard object is selected from horse flesh red eggs
White Apomyoglobin (equine), molecular weight 16952Da, sequence are SEQ ID NO:4：
GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILK
KKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG。
Also in other embodiments, the internal standard object is selected from any combination in above-mentioned average molecular weight range.
In one specific embodiment, wherein the internal standard object is selected from peptide material P (33507-63-0) and h-Mb
The combination of Apomyoglobin (equine).In another embodiment, the internal standard object is selected from P14R
The combination of (synthetic peptide) and h-Mb Apomyoglobin (equine)；In other specific embodiment parties
In case, the internal standard object is selected from ACTH fragment18-39 (human) and h-Mb Apomyoglobin
(equine) combination；And in other specific embodiment, the internal standard object is selected from selected from peptide material P
The combination of (33507-63-0) and ACTH fragment18-39 (human).
3rd purpose of the invention is to provide a kind of reagent for flight time mass spectrum microbiological specimens pre-treatment and combines
Object, main component include：Component I：Acetonitrile and formic acid；Component II：Acetonitrile, trifluoroacetic acid and alpha-cyano -4- hydroxycinnamic acids；
Component III：Internal standard object described in any of the above-described part.
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：Peptide material P (33507-63-0) (average molecular weight 1347.3Da).
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：P14(average molecular weight is R (synthetic peptide)
1533.85Da)。
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：(average molecular weight is ACTH fragment18-39 (human)
2465.19Da)。
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：(average molecular weight is h-Mb Apomyoglobin (equine)
16952Da)。
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：Peptide material P (33507-63-0) (average molecular weight 1347.3Da),
H-Mb Apomyoglobin (equine) (average molecular weight 16952Da).
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：P14(average molecular weight is R (synthetic peptide)
1533.85Da), h-Mb Apomyoglobin (equine) (average molecular weight 16952Da).
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：(average molecular weight is ACTH fragment18-39 (human)
2465.19Da), h-Mb Apomyoglobin (equine) (average molecular weight 16952Da).
In one embodiment, wherein component I includes：The formic acid of the acetonitrile of 50.0% (v/v), 35.0% (v/v), it is remaining
It measures as water.Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/v) alpha-cyano-
4- hydroxycinnamic acids, surplus are water.Component III includes：Peptide material P (33507-63-0) (average molecular weight 1347.3Da),
ACTH fragment18-39 (human) (average molecular weight 2465.19Da).
4th purpose of the invention is to provide by being used for mass spectrum prepared by above-mentioned internal standard object or reagent composition
Identify the detection product of unknown microorganism.
In one embodiment, which detects the kit of microorganism for flight time mass spectrum, including：
(1) reagent composition of microbiological specimens pre-treatment；
(2) internal standard compositions；
In one embodiment, which further includes：(3) other reagents for mass spectrogram, including negative quality-control product, positive matter
Control product.
In another embodiment, which further includes point sample and Mass Spectrometer Method target piece and for comparing and school
The software of positive reference substance and determinand molecular weight.
The 5th purpose of the present invention is to provide by by above-mentioned internal standard object or reagent composition or detection product
For the Internal standard correction methods method of flight time mass spectrum system identification microbiological specimens, step includes：
(1) centrifugation equipped with 10 μ l components I is fallen with sterilizing toothpick (or 1 μ l oeses) suitable single bacterium in picking part
Guan Zhong carries out clasmatosis, albumen and polypeptide release, cracks 5 minutes.
(2) above-mentioned 1 μ l of solution is taken to be added on the hole position of target plate, drying at room temperature.
(3) 1 μ l of internal standard object is taken to cover on same hole position, are spontaneously dried；
(4) micro-pipe of component II is opened, 1 μ l components II is pipetted and is added on same hole position, spontaneously dry.
(5) target plate is put into flight time mass spectrum system and is detected, wherein to be measured point by comparing internal standard object
Son amount is divided with actual
The difference of son amount, is corrected for the molecular weight of tested microorganism characteristic protein.
In one embodiment, the microorganism detection is to determine the genus and species, subspecies or hypotype of microorganism.At one
In specific embodiment, the microorganism detection be non-diagnostic purpose detect pathogenic bacteria, contaminated bacteria, drug-fast bacteria etc. or have
The pathogenic bacteria of diagnostic purpose.
In one embodiment, the microorganism in any of the above-described scheme is microorganism in environmental pollution, food quarantine
In microorganism, the microorganism in import-export commodity, resistant micro-organism etc. in drug research.
In one embodiment, the microorganism in any of the above-described scheme is prokaryotic micro-organisms, eukaryotic microorganisms.At one
In specific embodiment, the prokaryotic micro-organisms includes bacterium.The eukaryotic microorganisms include saccharomycete, mould etc. for fungi.
Principle and definition
The principle of flight time mass spectrum system identification microorganism is：Different microorganisms, the protein contained is variant,
After processing, using flight time mass spectrum system detectio, different microorganisms has its different characteristic fingerprint to microbiological specimens
Collection of illustrative plates is compared by flight time mass spectrum network analysis software with the feature spectrogram of known microorganisms in database,
Not same species of microorganism is distinguished, that is, provides the qualification result of microorganism.Since the characteristic fingerprint spectrogram of microorganism is flight time matter
Spectra system identifies the unique basis for estimation of microorganism, thus spectrogram is accurately the correct premise of microbial identification.Each single
Standard substance is added in sample to be tested, after being corrected by analysis software to single sample, then microbial identification is carried out, so as to improve
The accuracy rate of single sample detection.
Although it should be pointed out that the present invention can be used for detect microorganism, the present invention be only to testing result into
Row correction, so that testing result is consistent with actual result.Due to by flight time mass spectrum system detectio microorganism, it is necessary to
The characteristic protein collection of illustrative plates of previously prepared tested microorganism, the inspection can not be completed by relying solely on the internal standard object of the present invention
It surveys, therefore detection method according to the present invention and the diagnosis or detection that are not belonging to disease.
Technique effect
(1) present invention adds standard substance by research in micro-biological samples, it is found that the addition of internal standard material can
Single microbial sample is corrected, and is used in combination with flight time mass spectrum system micro-biological samples reagent treatment, can be carried
The accuracy rate of high microorganism identification, while compensate for and only have external standards to do the deficiency corrected currently on the market；
(2) molecular weight ranges of internal standard material that the present invention is added are beyond microbial identification scope, and
Within detection range, the requirement of detection and correction can reach；
(3) it is applicable to include using the method for internal standard material correction single microbial sample detection in the present invention
In all kinds of microbiological specimens for MALDI-TOF MS detections such as fungi, bacterium.
Description of the drawings
Fig. 1-1：Kirschner citric acid bacillus Citrobacter koseri
Fig. 1-2：Escherichia coli Escherichia coli
Fig. 1-3：Enterococcus faecalis Enterococcus faecalis
Fig. 1-4：Friedlander's bacillus Klebsiella pneumoniae
Fig. 1-5：Stenotrophomonas maltophilia Stenotrophomonas maltophilia
Fig. 1-6：Pseudomonas aeruginosa Pseudomonas aeruginosa
Fig. 1-7：Salmonella Salmonella sp.
Fig. 1-8：Klebsiella oxytoca Klebsiella oxytoca
Fig. 1-9：Human fetal cardiomyocytes Staphylococcus hominis
Fig. 1-10：Acinetobacter baumannii Acinetobacter baumannii
Fig. 1-11：Enterobacter cloacae Enterobacter cloacae
Fig. 1-12：Serratia marcescens Serratia marcescens
Fig. 2-1：Utilize trifluoroacetic acid/acetonitrile organic solvents dissolving polypeptide mass spectrogram
Fig. 2-2：Utilize ultrapure water dissolution polypeptide mass spectrogram
Fig. 2-3：Utilize ultrapure water dissolution internal standard composition
Fig. 2-4：Utilize trifluoroacetic acid/acetonitrile dissolving internal standard composition
Fig. 3-1：Internal standard is added using patter method processing micro-biological samples Escherichia coli (ATCC8739)
Fig. 3-2：It adds internal standard and utilizes a step treatment by extraction micro-biological samples Escherichia coli (ATCC8739)
Fig. 3-3：Internal standard is added using three step centrifugal process processing micro-biological samples Escherichia coli (ATCC8739)
Fig. 3-4：Mixing addition internal standard composition (internal standard compound pulls open figure)
Fig. 3-5：Mixing addition internal standard composition figure
Fig. 4-1：There is the peak of deviation using patter method processing micro-biological samples Escherichia coli (ATCC8739) in addition internal standard
Figure
Fig. 4-2：There is deviation again using patter method processing micro-biological samples Escherichia coli (ATCC8739) in addition internal standard
To the peak figure after instrumental correction
Fig. 5：There is obvious characteristic peak figure at m/z1351
Fig. 6-1：Staphylococcus haemolyticus
Fig. 6-2：Staphylococcus aureus
Fig. 6-3：Human fetal cardiomyocytes
Fig. 6-4：Pseudomonas aeruginosa
Fig. 6-5：Proteus vulgaris
Fig. 6-6：Klebsiella oxytoca
Fig. 6-7：Acinetobacter bauamnnii
Fig. 6-8：Streptococcusagalactiae
Fig. 6-9：Enterococcus faecium
Fig. 7：Staphylococcus epidermis
Fig. 8：Aeromonas hydrophila
Fig. 9：Utilize the protein fingerprint spectrum of the ultrapure water dissolution identification Escherichia coli of h-Mb standard substance
Specific embodiment
In order to further appreciate that the technical characteristic of the present invention, detailed explain is carried out to the present invention with reference to specific embodiment
It states.Embodiment only has the present invention illustrative effect, without the effect of any restrictions, those skilled in the art
The modification of any unsubstantiality is made on the basis of the present invention, should all be belonged to the scope of protection of the present invention.
Embodiment one utilizes flight time mass spectrum system micro-biological samples pretreatment reagent kit identification microorganism
Strain culturing and pre-treatment：37 DEG C of cultures 24 of Clinical isolation that more plants of Beijing hospital laboratories are preserved are small
When, corresponding single bacterium colony is obtained, target plate is applied to using sterilizing toothpick (or 1 μ l oeses) suitable single bacterium colony in picking part
Corresponding points position is drawn 1 μ l components I and is covered on the point position, spontaneously dries, and draws 1 μ l components II and covers on same hole position, from
So after drying, upper machine testing and identification and analysis, qualification result such as table 1 and Fig. 1-1 to Fig. 1-12.
The qualification result of 5 plants of bacterium in 1 embodiment one of table
| Bacterium numbering | Strain Chinese | Strain latin name |
| 1-1 | Kirschner citric acid bacillus | Citrobacter koseri |
| 1-2 | Escherichia coli | Escherichia coli |
| 1-3 | Enterococcus faecalis | Enterococcus faecalis |
| 1-4 | Friedlander's bacillus | Klebsiella pneumoniae |
| 1-5 | Stenotrophomonas maltophilia | Stenotrophomonas maltophilia |
| 1-6 | Pseudomonas aeruginosa | Pseudomonas aeruginosa |
| 1-7 | Salmonella | Salmonella sp. |
| 1-8 | Klebsiella oxytoca | Klebsiella oxytoca |
| 1-9 | Human fetal cardiomyocytes | Staphylococcus hominis |
| 1-10 | Acinetobacter baumannii | Acinetobacter baumannii |
| 1-11 | Enterobacter cloacae | Enterobacter cloacae |
| 1-12 | Serratia marcescens | Serratia marcescens |
From above-mentioned qualification result, principal character peak is distributed in microbial biomass spectrogram (see Fig. 1-1 to Fig. 1-12)
Between 2000-13000Da, therefore, the internal standard object molecular weight added in the present invention is less than 2000Da or/and is more than
During 13000Da, the similar mass spectra peak of microorganism characteristic peak will not be resulted from, so as to theoretically ensure that the feasible of microbial identification
Property and accuracy.
It is prepared by the internal standard composition of two flight time mass spectrum system identification microorganism of embodiment
(1) trifluoroacetic acid/acetonitrile dissolving standard substance is utilized
Using trifluoroacetic acid/acetonitrile organic solvents (0.1% trifluoroacetic acid and 10% acetonitrile) by polypeptide standard items dry powder (i.e.
Polypeptide P (33507-63-0), molecular weight 1347Da) solution of 100fmol/ul concentration is dissolved into, 1 μ l are drawn after careful mixing and are covered
It covers on target plate point position, spontaneously dries, it is rear to draw component in the commercially available flight time mass spectrum system micro-biological samples reagent treatments of 1 μ l
II (or other for the matrix liquid in microbial identification) covers same point position, after natural drying, time of-flight mass spectrometer
It is detected, after instrumental correction, there is apparent spy in testing result such as Fig. 2-1 of the standard substance at m/z1348.76
Peak is levied, illustrates that the standard substance can use trifluoroacetic acid/acetonitrile to dissolve, and with good detection result.
(2) ultrapure water dissolution standard substance is utilized
Polypeptide standard items dry powder (i.e. polypeptide P (33507-63-0), molecular weight 1347Da) is dissolved into using ultra-pure water
The solution of 500fmol/ul concentration is drawn 1 μ l after careful mixing and is covered on target plate point position, and after natural drying, it is commercially available to draw 1 μ l
Compositionⅱ in flight time mass spectrum system micro-biological samples reagent treatment (or other for the matrix liquid in microbial identification) covers
It covers on same point position, after natural drying, time of-flight mass spectrometer is detected, testing result such as Fig. 2-2, in m/
There is apparent characteristic peak at z1348.88, illustrate that the standard substance can use ultrapure water dissolution, and with good detection result.
(3) ultrapure water dissolution internal standard composition is utilized
Peptide material (i.e. polypeptide P (33507-63-0), the molecular weight of final concentration of 500fmol/ μ L is prepared with ultra-pure water
1347Da) and the mixed solution of the P14R (molecular weight 1533) of final concentration of 50fmol/ μ L.1 μ l coverings are drawn after careful mixing
Onto target plate point position, spontaneously dry, it is rear to draw compositionⅱ in the commercially available flight time mass spectrum system micro-biological samples reagent treatments of 1 μ l
(or other for the matrix liquid in microbial identification) covers same point position, after natural drying, time of-flight mass spectrometer into
Row detection, after instrumental correction, testing result such as Fig. 2-3 of the internal standard composition, in m/z 1348.64 and m/z
There is apparent characteristic peak at 1534.65, illustrate that the internal standard composition can use ultrapure water dissolution, and with good detection result.
(4) trifluoroacetic acid/acetonitrile dissolving internal standard composition is utilized
It is prepared using trifluoroacetic acid/acetonitrile organic solvents (0.1% trifluoroacetic acid and 10% acetonitrile) final concentration of
The mixing of the P14R (molecular weight 1533) of the peptide material (molecular weight 1347Da) of 500fmol/ μ L and final concentration of 50fmol/ μ L
Solution.1 μ l are drawn after careful mixing to cover on target plate point position, are spontaneously dried, it is rear to draw the commercially available flight time mass spectrum systems of 1 μ l
Compositionⅱ in micro-biological samples reagent treatment (or other for the matrix liquid in microbial identification) covers same point position, from
So after drying, time of-flight mass spectrometer is detected.After instrumental correction, testing result such as Fig. 2-4 of the internal standard composition,
There is apparent characteristic peak at m/z 1348.48 and m/z1534.48, illustrate that the internal standard composition can have with trifluoroacetic acid/acetonitrile
Solvent dissolves, and with good detection result.
As above described in figure, the theoretical molecular weight (1347.63Da) of internal standard object polypeptide P (33507-63-0) and P14R's
Theoretical molecular weight (1533.85Da), with actually detected molecular weight there are subtle difference, and same internal standard object is (such as
Polypeptide P) each detected value there is also certain difference, this explanation is in the molecular weight mistake of laser mass spectrometry detection microorganism characteristic protein
Unavoidably there are systematic errors in journey.Therefore, in actually detected microbial process, by adding in above-mentioned single or joint
Ministerial standard object corrects systematic error, so as to more accurately detect and distinguish different microorganisms and its close molecular weight
Characteristic protein.
The internal standard composition adding method optimization of three flight time mass spectrum system identification microorganism of embodiment
(1) internal standard composition is directly added
Using patter method processing micro-biological samples Escherichia coli (ATCC8739), and internal standard composition is directly appended to sample
On this point position, concrete operation method is：Sterile toothpick picking Escherichia coli single bacterium colony is uniformly applied on target plate point position, natural
After drying, 1 μ l internal standards composition is covered on same point position and (or the commercially available flight time mass spectrum systems of 1 μ l are covered in sample spot
Components I in micro-biological samples reagent treatment covers 1 μ l internal standards composition after natural drying), it spontaneously dries, it is commercially available winged to draw 1 μ l
Compositionⅱ (or other are used for the matrix liquid in microbial identification) covering in row time mass spectrum system micro-biological samples reagent treatment
Onto same point position, time of-flight mass spectrometer detection is carried out after natural drying.After instrumental correction, the testing result of the sample is such as
There is apparent characteristic peak in Fig. 3-1 at m/z1348, and when illustrating using patter method processing microbiological specimens, internal standard composition can
It is added directly into sample spot and detects.
Using a step treatment by extraction micro-biological samples Escherichia coli (ATCC8739), and internal standard composition is directly added
Onto sample point position, concrete operation method is：The commercially available flight time mass spectrum system microorganisms of 30 μ l are added in 200 μ l centrifuge tubes
Component I in sample processing reagent is fallen with 1 μ l aseptic inoculations rings or pipette tips picking single bacterium in upper tube, is shaken mixing 5min, is added 1 μ
L samples spontaneously dry on target plate, 1 μ l internal standard compositions are covered on same point position, spontaneously dry, and draw the 1 commercially available flights of μ l
Compositionⅱ in time mass spectrum system micro-biological samples reagent treatment (or other for the matrix liquid in microbial identification) covers
On same point position, time of-flight mass spectrometer detection is carried out after natural drying.After instrumental correction, the testing result of the sample is as schemed
3-2 has apparent characteristic peak at m/z1348, when illustrating using a step treatment by extraction microbiological specimens, internal standard composition
It can be added directly into sample spot and detect.
Using three step centrifugal process processing micro-biological samples Escherichia coli (ATCC8739), and internal standard composition is directly added
Onto sample point position, concrete operation method is：300 μ l pure water are added in 1.5ml or 2ml centrifuge tubes, oese takes large intestine bar
Bacterium single bacterium is fallen in upper tube, and concussion is uniform, adds in 900 μ l ethyl alcohol, concussion, and 12000rpm is centrifuged 2 minutes, supernatant discarding, centrifugation 2
Minute, it spontaneously drying, adds 50 μ l70% formic acid, fully shaking adds 50 μ l acetonitriles, fully shaking, and 12000rpm is centrifuged 2 minutes,
Add 1 μ l samples to target plate, spontaneously dry, 1 μ l internal standard compositions are covered on same point position, spontaneously dry, it is commercially available to draw 1 μ l
Compositionⅱ in flight time mass spectrum system micro-biological samples reagent treatment (or other for the matrix liquid in microbial identification) covers
It covers on same point position, time of-flight mass spectrometer detection is carried out after natural drying.After instrumental correction, the testing result of the sample
Such as Fig. 3-3, there is apparent characteristic peak at m/z1348, when illustrating using three step centrifugal process processing microbiological specimens, internal standard group
Conjunction object, which can be added directly into sample spot, to be detected.
(2) mixing addition internal standard composition
1 is pressed using patter method processing micro-biological samples Escherichia coli (ATCC8739), and internal standard composition and matrix liquid:
The mixing of 1 ratio is added on sample point position, and concrete operation method is：Sterile toothpick picking Escherichia coli single bacterium colony is uniformly smeared
On target plate point position, after natural drying, 1 μ l internal standards compositions are covered on same point position and commercially available flight time mass spectrum system is micro-
The mixture of compositionⅱ (or other are used for the matrix liquid in microbial identification), spontaneously dries laggard in biological sample reagent treatment
Row time of-flight mass spectrometer detects.After instrumental correction, testing result such as Fig. 3-4 and 3-5 of the sample, in m/z1348 and
There is apparent characteristic peak at m/z1534, when illustrating using patter method processing microbiological specimens, internal standard composition can be mixed with matrix liquid
Conjunction is added in sample spot and detects.
The internal standard composition calibration result evaluation of example IV flight time mass spectrum system identification microorganism
Using the mass spectrogram of internal standard composition Caliberation Flight time mass spectrum system detectio microorganism, by Escherichia coli
(ATCC8739) it is inoculated on Columbia Blood Agar culture medium, when 37 DEG C of cultures 24 are small, sterile toothpick picking Escherichia coli are single
Bacterium colony is uniformly applied on target plate point position, and after natural drying, 1 μ l internal standards composition is covered on same point position (or in sample spot
Components I in the upper covering commercially available flight time mass spectrum system micro-biological samples reagent treatments of 1 μ l, covers 1 μ l internal standards after natural drying
Composition), spontaneously dry, draw the commercially available flight time mass spectrum system micro-biological samples reagent treatments of 1 μ l in compositionⅱ (or other
For the matrix liquid in microbial identification) same point position is covered, time of-flight mass spectrometer detection is carried out after natural drying, it should
The testing result of sample such as Fig. 4-1 without obvious characteristic peak at m/z1348, illustrates that deviation occurs in mass spectra peak in the mass spectrogram,
The Mass Spectrometer Method result of the sample is insincere, need to re-start instrumental correction and gathered data.
Above-mentioned E. coli SampLes judge that deviation occurs in mass spectrogram, again right through internal standard composition characteristic peak m/z1348 values
After instrumental correction, mass spectrometric data is gathered, there are obvious characteristic peak, explanation in testing result such as Fig. 4-2 in the mass spectrogram at m/z 1348
The mass spectra peak of the mass spectrogram does not occur deviation, the Mass Spectrometer Method credible result of the sample.
The internal standard composition calibration result evaluation of five flight time mass spectrum system identification microorganism of embodiment
Using the mass spectrogram of internal standard composition Caliberation Flight time mass spectrum system detectio microorganism, by Escherichia coli
(ATCC8739) it is inoculated on Columbia Blood Agar culture medium, when 37 DEG C of cultures 24 are small, sterile toothpick picking Escherichia coli are single
Bacterium colony is uniformly applied on target plate point position, and after natural drying, 1 μ l internal standards composition is covered on same point position (or in sample spot
Components I in the upper covering commercially available flight time mass spectrum system micro-biological samples reagent treatments of 1 μ l, covers 1 μ l internal standards after natural drying
Composition), spontaneously dry, draw the commercially available flight time mass spectrum system micro-biological samples reagent treatments of 1 μ l in compositionⅱ (or other
For the matrix liquid in microbial identification) same point position is covered, time of-flight mass spectrometer detection is carried out after natural drying, it should
The testing result of sample such as Fig. 5, there is apparent characteristic peak at m/z1351, and does not have obvious characteristic peak at m/z1348,
Illustrate that deviation occurs in mass spectra peak in the mass spectrogram, which is identified using the identification software containing calibration function, identify
As a result it is Escherichia coli, and its corresponding confidence level score value is 94, illustrates that the internal standard has good calibration result.
Embodiment six utilizes flight time mass spectrum system micro-biological samples reagent treatment and the internal standard composition in the present invention
Detect micro-biological samples
Clinical common pathogenic bacteria (table 2) is detected according to the detection of five the method for embodiment, testing result such as Fig. 6-1
To 6-9, qualification result is table 3 after software analyzes and processes.
2 clinical common pathogenic bacteria list of table
3 clinical common pathogenic bacteria qualification result of table
| Bacterium numbering | Strain latin name | Strain Chinese name | Qualification result |
| 6-1 | Staphylococcus haemolyticus | Staphylococcus haemolyticus | 93 |
| 6-2 | Staphylococcus aureus | Staphylococcus aureus | 96 |
| 6-3 | Staphylococcus hominis | Human fetal cardiomyocytes | 96 |
| 6-4 | Pseudomonas aeruginosa | Pseudomonas aeruginosa | 127 |
| 6-5 | Proteus vulgaris | Proteus vulgaris | 130 |
| 6-6 | Klebsiella oxytoca | Klebsiella oxytoca | 93 |
| 6-7 | Acinetobacter baumannii | Acinetobacter bauamnnii | 119 |
| 6-8 | Streptococcus agalactiae | Streptococcusagalactiae | 120 |
| 6-9 | Enterococcus faecium | Enterococcus faecium | 124 |
Embodiment seven utilizes flight time mass spectrum system micro-biological samples reagent treatment and the internal standard composition in the present invention
Detect the pathogenic bacteria of skin infection patient
Suppuration sexual secretion sample at the surgical site infection patient wound of certain hospital is subjected to inoculated and cultured, is obtained doubtful
After the single bacterium colony of pathogenic bacteria, carry out micro-biological samples processing using five the method for embodiment and examined using time of-flight mass spectrometer
It surveys, obtained testing result such as Fig. 7, qualification result is the staphylococcus epidermis result and full-automatic biochemical assessing instrument qualification result
Unanimously.
Embodiment eight utilizes flight time mass spectrum system micro-biological samples reagent treatment and the internal standard composition in the present invention
Detect the pathogenic bacteria in food
The results are shown in Figure 8 for Mass Spectrometric Identification.
Embodiment nine utilizes h-Mb standard substance identification Escherichia coli
Method：Ultrapure water dissolution, the results are shown in Figure 9 for Mass Spectrometric Identification.
Claims (2)
1. a kind of reagent composition for flight time mass spectrum microbiological specimens pre-treatment, main component includes：Component I：
Acetonitrile and formic acid；Component II：Acetonitrile, trifluoroacetic acid and alpha-cyano -4- hydroxycinnamic acids；Component III：1000Da<Average molecular weight
<3000Da and 12000Da<Average molecular weight<The internal standard object of 20000Da,
Wherein described internal standard object is selected from 1000Da<Average molecular weight<The polypeptide P (33507-63-0) of 2000Da and/or more
Peptide P14R(synthetic peptide)；And/or
Wherein described internal standard object is selected from 2000Da<Average molecular weight<The polypeptide A CTH fragment18-39 of 3000Da
(human)；
And wherein the internal standard object is selected from 12000Da<Average molecular weight<The h-Mb of 180000Da
Apomyoglobin(equine)。
2. reagent composition as described in claim 1, wherein component I includes：The acetonitrile of 50.0% (v/v), 35.0% (v/v)
Formic acid, surplus is water；Component II includes：The acetonitrile of 52.5% (v/v), the trifluoroacetic acid of 2.5% (v/v), 15mg/ml (m/
V) alpha-cyano -4- hydroxycinnamic acids, surplus are water.
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| CN201810345986.4A CN108519267B (en) | 2016-11-25 | 2016-11-25 | Kit for detecting microorganisms by internal standard substance spectrum |
| CN201810345905.0A CN108802162B (en) | 2016-11-25 | 2016-11-25 | Method for detecting microorganisms by internal standard substance spectrum |
| CN201611062751.1A CN107024530B (en) | 2016-11-25 | 2016-11-25 | Method that detection microorganism is composed by internal standard material and products thereof |
| CN201810346086.1A CN108593753B (en) | 2016-11-25 | 2016-11-25 | Internal standard correction method for detecting microorganisms through internal standard substance spectrum |
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| CN201810346086.1A Division CN108593753B (en) | 2016-11-25 | 2016-11-25 | Internal standard correction method for detecting microorganisms through internal standard substance spectrum |
| CN201810345905.0A Division CN108802162B (en) | 2016-11-25 | 2016-11-25 | Method for detecting microorganisms by internal standard substance spectrum |
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| CN108085421B (en) * | 2018-01-24 | 2018-12-04 | 北京毅新博创生物科技有限公司 | The method and products thereof of mass spectrography detection influenza A virus multiple PCR products |
| CN109900774B (en) * | 2018-12-13 | 2020-07-28 | 中国科学院生态环境研究中心 | Method for improving detection sensitivity of MA L DI-TOF-MS to PS micro/nano particles |
| CN109884160A (en) * | 2019-02-26 | 2019-06-14 | 中国矿业大学 | Using the method for mode identification technology identification Carbapenem-resistant paracolon |
| TWI700492B (en) * | 2019-09-17 | 2020-08-01 | 長庚大學 | Molding characteristic mass spectrum and identification model establishment method and method of analysis and identification of microbial characterization |
| CN111876343A (en) * | 2020-06-24 | 2020-11-03 | 四川省农业科学院分析测试中心 | Mass spectrometry method for detecting Klebsiella pneumoniae in instant fresh fruits and vegetables |
| CN112540139B (en) * | 2020-12-16 | 2022-07-15 | 杭州汇健科技有限公司 | Molecular weight calibrator kit for metabolic profiling detection, and preparation method and use method thereof |
| CN113933377A (en) * | 2021-09-28 | 2022-01-14 | 深圳湾实验室 | Compound and mass spectrum standard substance and calibration substance thereof |
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| CN108593753A (en) | 2018-09-28 |
| CN107024530A (en) | 2017-08-08 |
| CN108519267B (en) | 2020-06-05 |
| CN108519267A (en) | 2018-09-11 |
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