CN107024370A

CN107024370A - A kind of kit of flight time mass spectrum system micro-biological samples pre-treatment

Info

Publication number: CN107024370A
Application number: CN201610696197.6A
Authority: CN
Inventors: 马庆伟; 冯立平; 王杰; 游胜群
Original assignee: Beijing Yixin Bochuang Biological Technology Co Ltd
Current assignee: Beijing Yixin Bochuang Biological Technology Co Ltd
Priority date: 2016-08-20
Filing date: 2016-08-20
Publication date: 2017-08-08
Anticipated expiration: 2036-08-20
Also published as: CN108507845B; CN107024370B; CN108507845A

Abstract

The invention discloses a kind of reagent composition for flight time mass spectrum system micro-biological samples pre-treatment, said composition can effectively crack bacterium, and the progress Mass Spectrometer Method using total protein as target body.The reagent composition can obtain the characteristic peak spectrogram that signal is strong, discrimination is high, and have the more reliable and stable degree of accuracy for final identification fraction.The present invention also includes the mass spectrometry kit of reagent composition, and detects the preprocess method and detection method of micro-biological samples.

Description

Kit for pretreatment of microbial sample of time-of-flight mass spectrometry system

Technical Field

The invention belongs to the technical field of biology, and relates to a kit and a detection product for pretreating a microorganism sample by a time-of-flight mass spectrometry system.

Background

Time of Flight Mass Spectrometer (TOF), a very common Mass Spectrometer whose Mass analyzer is an ion drift tube (ion drift tube) where ions generated by an ion source are first collected, all the ions in the collector become 0, accelerated using a pulsed electric field into a field-free drift tube and fly at a constant velocity towards an ion receiver, the larger the Mass of the ions, the longer it takes to reach the receiver; the smaller the mass of the ions, the shorter the time it takes to reach the receiver, and according to this principle, ions of different masses can be separated in the size of m/z. The flight time mass spectrometer has the advantages of large detectable molecular weight range, high scanning speed and simple instrument structure.

The accuracy rate of the time-of-flight mass spectrometry for completing the detection of the microbial sample can reach 99.9%, and the method has the advantages of high accuracy, strong flexibility, large flux, short detection period and the like, and is most attractive and should also have the cost performance. The time-of-flight mass spectrometry platform (MALDI-TOF) is an international universal research platform for gene Single Nucleotide Polymorphism (SNP), and the method has become a gold standard in the field by virtue of the scientificity and accuracy of the method.

Microorganisms are various in types and complex in composition, and identification results thereof vary depending on growth sites, growth conditions and detection means: according to the growth characteristics of microorganisms, different modes and conditions need to be cultured, and the primary identification is carried out according to the colony morphology and the gram stain; the variation of the microorganism can cause the continuous change of biochemical reaction, and the bacteria with atypical biochemical reaction are difficult to identify; the nucleic acid detection method as the gold standard has accurate and reliable result, but has high technical requirement, time consumption and special operators, and the method has complicated steps and long detection period and cannot timely and effectively guide the application and treatment of antibiotics. Extensive clinical empirical medication leads to an increase in drug-resistant strains, further increasing the difficulty of clinical treatment. Rapid identification of clinical microorganisms has become an urgent problem to be solved. With the development of scientific technology, the processes of dyeing, inoculation culture, biochemistry, serology, drug sensitivity experiment and the like in the microorganism identification are automated to different degrees, and the identification efficiency is greatly improved. In addition, based on the development of molecular biology technology, many methods for rapidly identifying pathogenic microorganisms appear, such as real-time quantitative polymerase chain reaction, restriction fragment length polymorphism analysis, single-strand conformation polymorphism analysis, pyrosequencing technology, fluorescence in situ hybridization, gene chip, etc., for example, Lixiaxia et al (reference 1: medical review, volume 17, phase 13, 7 months 2011) report that the advantages and disadvantages of various detection technologies in clinical application are introduced in the application and research progress of clinical pathogenic bacteria rapid identification technology, and the methods do not need to carry out complex operations of culture separation and biochemical identification on clinical microorganisms, have the characteristics of strong specificity, high sensitivity, good repeatability, high throughput, diversity, miniaturization, automation, etc., and have high requirements on fragment specificity, length limitation and technology required for detecting different bacteria, the spread in clinical microbiological routine is limited.

The method for identifying the microorganisms by using the mass spectrometry technology has the obvious advantages that: (1) the sample is processed simply in the early stage; (2) the operation is simple and rapid, and the flux is high; (3) the sensitivity is high; (4) the accuracy is good. The sample can be directly loaded through a simple protein extraction step, a map is obtained through matrix-assisted laser desorption ionization flight time, and then the obtained map is compared with species/genus specific maps of different microorganism families in a database, so that an identification result can be obtained, and the method is efficient, rapid, low in price and good in clinical application potential. However, Li Xiaoxia et al (2011) also indicate that although TOF MS mass spectrometry can accurately identify pathogenic bacteria and provide key treatment information for infectious diseases, false negative conditions exist, the result may be related to that the content of bacteria in blood culture solution is too low to form a specific recognizable peak, and protein components in blood interfere with the formation of mass spectrometry to a certain extent, so that the accuracy of the identification result is influenced.

Furthermore, in TOF MS techniques, the choice of matrix directly affects the analytical results of the sample. To date, the choice of effective matrix has not been fully theorized and has been obtained only by experimental screening of a large number of compounds. In general, the substrates used in TOFMS are organic acidic compounds, such as cinnamic acid derivatives HCCA and SA, aromatic carboxylic acid derivative 2, 5-dihydroxybenzoic acid (DHB), which are highly efficient substrates for analyzing polypeptides and proteins, and 3-hydroxypicolinic acid (3-HPA) and Picolinic Acid (PA), which are commonly used substrates for DNA analysis, such as XuXinRong et al (reference 2: "Proc. Natl. Acad. Sci. 27, vol. 3, 2011) which report the molecular mass distribution of hydrolyzed collagen protein powder in positive ion mode using α -cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid and sinapic acid, respectively, as substrates. The analysis results show that: sinapic acid is the most suitable substrate, and the analysis condition is the combination of a linear mode and a reflection mode, so that the molecular weight distribution of the hydrolyzed collagen powder can be accurately and quickly determined. The method is superior to other conventional molecular weight determination methods.

As another example of the substrate for analyzing saccharides, 2, 5-dihydroxybenzoic acid (2, 5-DHB), Sinapinic Acid (SA), T-CN-4-hydroxycinnamic Acid (ACHC) and the like are available, for example, Helmink et al (reference 3: Proc. Natl. Acad. Hippocampus, 1998, 7 months) have found a substrate 2,5-DHB suitable for saccharide analysis by comparing the analytical effects of 3 commonly used substrates MALDI-MS of saccharide compounds and the MALDI-MS spectra of oligosaccharide and polysaccharide positive and negative ions, and have studied the process of forming ions under the conditions of laser desorption/ionization of saccharide compounds, and pointed out the important role of N a + and K + ions in the measurement of oligosaccharide molecules, and the molecular weight of glucan having a molecular weight of more than 10000 has been successfully determined by means of column chromatography separation. However, it is not necessary that the compound be acidic and be effective as a substrate, and certain basic compounds may also be useful as substrates. For example, basic substrates reported by Zhou Li Hua et al (reference 4: Proc. Natl. Acad. Sci. higher school Chemicals, 2004, 12.P.) for analyzing polypeptides, proteins and DNAs include 2-amino-4-methyl-5-nitropyridine, 2-amino-5-nitropyridine, p-nitroaniline, 9-aminoacridine and the like. The use of a basic matrix can further expand the scope of application of TOF MS, particularly for the determination of acid sensitive substances.

At present, in TOF MS techniques for detecting microorganisms, the following substrates are commonly used: alpha-cyano-4-hydroxycinnamic acid (CHCA), 2, 5-dihydroxybenzoic acid (2, 5-dihydrobenzoic acid, DHB), 2-amino-4-methyl-5-nitropyridine (2-amino-4-methyl-5-nitropyridine, 2a4m5n), trans-3-indoleacrylic acid (IAA), 2-mercaptobenzothiophene (2-mercaptobenzothiazole, MBT), 2- (4-hydroxyimine) benzoic acid [2- (4-hydroxyheterocyclic) benzoic acid, HABA ], 2, 6-dihydroxybenzoic acid (2, 6-dihydroacetophenone, DHAP), sinapic acid (3, 5-dihydronaphthalene-4-dihydrothiophene, 5-mercaptobenzothiazole), 2-mercaptobenzothiazole (2 a 4-mercaptobenzothiazole, CMBT), for example, bright-day mushroom and the like (reference 5: "sanitation research" volume 37, phase 6, 2008) used a CHCA matrix for the detection and typing of 88 salmonella strains isolated from the stools of outpatient diarrhea patients using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS); lifengqin and the like (reference 6: Chinese food sanitation journal, volume 19, No. 5, 2007) screen matrix, matrix-sample ratio, sample loading method and the like used by MALDI-TOF-MS for detecting food fungi, obtain optimized experimental conditions, and research the reproducibility and practical application of the method. The results show that the MALDI-TOF-MS analysis has different optimal matrixes required by different fungi, and the optimal matrixes of monascus and fusarium are MBT; the most suitable substrates for Aspergillus, Penicillium and yeast are IAA; the optimal base/sample ratio for loading was 1: 1; the mixed liquid drop drying method is the best loading method. While the solvent used to formulate the above matrix is typically acetonitrile, trifluoroacetic acid (TFA) (bright-coloured chamois, lienfenqin), for example acetonitrile +0.1% aqueous TFA (70 + 30, volume fraction), or acetonitrile: TFA: water = 50: 2.5: 47.5 or 50: 0.1: 49.9.

in addition, for the most common microorganism pretreatment Kit VITEK Kit (MS-CHCAMATRIX for use with VITEK MS) commercially available on the market, the VITEK MS-CHCA matrix (yellow cap) reagent main components (required by both bacteria and fungi) are as follows: alpha-cyano-4-hydroxycinnamic acid, VITEK MS-FA (Red cover) reagent main component: 70% formic acid, when treated with this agent against fungi, and then using a matrix of VITEK MS-CHCA. The test kit has the defects that before a time-of-flight mass spectrometry system is used for identifying strains, when fungi or bacteria are uncertain, whether a VITEK MS-CHCA matrix is directly used or a combination of VITEK MS-FA and the VITEK MS-CHCA matrix is used cannot be determined.

For another example, a researcher who purchases a conventional microorganism identification mass spectrometer, such as Autoflex speed TOF/TOF (TOF mass spectrometer) or microflex LT, has two microorganism pretreatment methods, one is a method of directly coating a target plate, bacteria are directly coated on the target plate, and then a user-prepared microorganism sample pretreatment reagent is covered, wherein the reagent comprises the following main components and concentrations: 10mg/ml CHCA base, 50% acetonitrile, 47.5% water and 2.5% trifluoroacetic acid. However, this method is not sufficient for some fungal treatments, and does not effectively lyse the fungal cell wall, and is therefore not suitable for some pathogenic fungi. Another acetonitrile extraction method of formic acid is used for treating strains, and has the disadvantages of multiple steps (3 times of centrifugation), and long time consumption (about 30 min). For this purpose, the acetonitrile formate extraction method used in evaluation of the ability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify clinical bacteria was evaluated by using a high-crystal or the like (reference 10: electronic journal of clinical laboratory management, volume 3, phase 1, 2015): placing a single colony in a 1.5ml Eppendorf tube, adding 300 mul of deionized water and 900 mul of absolute ethyl alcohol, fully and uniformly mixing, centrifuging at 13000 r/min for 2min, and removing supernatant; centrifuging at 13000 r/min for 2min, and sucking and discarding the residual liquid; adding 50 μ l of 70% formic acid, shaking, adding equal amount of acetonitrile, shaking, centrifuging at 13000 r/min for 2min, adding 1 μ l of supernatant dropwise onto MALDI target plate, drying at room temperature, coating 1 μ l of matrix solution on the sample spot, and drying at room temperature; and (6) performing detection on the machine. The system has a detection rate slightly lower than that of a traditional instrument for common bacteria, is more complete in bacteria species identification library, and can be beneficial to detecting special and rare bacteria. Although the method selects more than or equal to 1.7000 points as the judgment score of the strain to be detected, the method has many steps, and partial results still fall into the end points of slightly less than 1.7000 points, 2.000 points and the like, and the results are difficult to be confirmed or need to be verified repeatedly, so that artificial uncontrollable factors are added, the experimental results are different due to the experience of testers, and the judgment score is not completely stable.

The result of TOF mass spectrometry for detecting microorganisms cannot reach 100% accuracy or the identification score has certain variation, which is brought by the principle of mass spectrometry for detecting proteins. Wherein,

protein distribution in bacteria in prokaryotic microorganisms:

(1) the cell wall contains a small amount of outer membrane protein, porin, lipoprotein and the like, and the periplasmic space contains hydrolytic enzymes such as protease, nuclease and the like; synthetases, such as peptidoglycan synthase; a binding protein; a receptor protein.

(2) The cell membrane contains 50% -70% of proteins, and the proteins are mainly transport integrin and enzymatic peripheral proteins.

(3) A cytoplasmic carboxylase comprising ribulose-1, 5-bisphosphate carboxylase; the ribosome contains 34 large subunit proteins and 21 small subunit proteins.

Fungal protein distribution in eukaryotic microorganisms:

(1) the cell wall contains a small amount of protein, structural protein embedded between glucan and mannose; enzymes having catalytic action, such as dextranase, mannanase, sucrase, alkaline phospholipase and esterase, etc.

(2) The protein content of the cytoplasmic membrane is very low, and the sterol is generally contained

(3) The nuclear envelope of the nucleus contains a fibrous layer protein; the chromatin contains histones; non-histones of enzymes involved in transcription by DNA replication.

(4) The cell matrix contains rich proteins such as enzyme and the like, and accounts for 25-50% of the total cell protein; the cytoskeleton consists of a plurality of proteins such as keratin and the like, and has the functions of supporting and moving.

(5) Ribosomes are composed of about 40% protein and 60% RNA, and contain 49 large subunit proteins and 33 small subunit proteins.

(6) Lysosomes contain more than 40 types of acid hydrolases, which are also proteins.

(7) One type of microbody, called the peroxisome, is an organelle containing one or several oxidases, surrounded by a single membrane.

(8) The inner mitochondrial membrane contains ATP synthase complex and 4 kinds of lipoprotein complexes.

(9) Other organelles such as chloroplasts, vacuoles, chitinase, hydrogenase, etc. contain some proteins.

Since the flight mass spectrometry of microorganisms is used to identify species, the size range generally used is just in the range of the high effect of ribosomal protein ionization, which can provide an accurate map with little influence from the growth conditions of microorganisms (reference 7: "test medicine and clinic", volume 9, stage 14, bang, 2012). Therefore, in the current practice OF successfully detecting bacteria and fungi by using time-OF-flight mass spectrometry, the stable expression OF ribosomal proteins in bacteria is detected, Abdessalamc. et al (JOURNAL OF CLINICAL MICROBIOGY, Apr. 2010, p. 1169-, in the research progress of matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology in rapid detection of pathogenic bacteria, the preparation method of the sample has obvious influence on the mass spectrum information of the sample, and in most cases, the pure bacterial colony grown by the culture medium can be directly identified, but the growth time of the bacterial colony is not more than 48 hours, otherwise, the ribosomal protein in the bacterial body is degraded, so that a characteristic peak with strong signal can not be obtained, and the false identification is generated. Certain pathogenic bacteria with tough cell walls, such as fungi, require protein extraction techniques to obtain sufficient quantities of ribosomal proteins to help increase the accuracy of identification. Among them, two commercially available pretreatment products for mass spectrometric detection, used in Abdessalam c. and king, are for the effective extraction of ribosomal proteins in bacteria, and on the analytical software associated with a commercially available mass spectrometer, the bacterial genus judgment standard I: 2.300-3.000 is divided into a complete and reliable identification to the seed level; judgment criterion II: 2.000 to 2.299 are classified into reliable identification to the genus level and possible identification to the species level; judgment standard III: 1.700-1.999 is divided into the possible identification of the genus level; a judgment standard IV: 0.000 to 1.699 were scored as non-confident (reference 10; reference 11: Giovanna Pultrano, Journal of Microbiological Methods 94 (2013) 262-. Therefore, whether the identified microorganisms can obtain stable and accurate identification scores for the time-of-flight mass spectrometry detection technology is crucial to the high-throughput and rapid identification of microorganisms clinically.

However, in order to ensure the accuracy of the identification score of microbial protein by flight time mass spectrometry, the existing research focuses on how to improve the separation and extraction of microbial ribosomal protein, and reports that the colony growth time used in the separation and extraction of microbial ribosomal protein is not more than 48 hours, otherwise, degradation of ribosomal protein is caused, and the identification accuracy is affected. Therefore, the existing related improvement ideas are how to rapidly and efficiently extract ribosomal proteins of bacteria to be detected and avoid the degradation of the ribosomal proteins, so that the ribosomal proteins are accurately used for time-of-flight mass spectrometry (invip, 2013). However, the inventors found that the identification scores of the mass spectrometric detection technique targeting ribosomal proteins and the improved technique thereof sometimes fluctuate among the above 4 criteria, and especially when the theoretical identification score should fall into the credible interval (criterion III) and the actual identification score falls into the untrustworthy interval (criterion IV), repeated detection is sometimes necessary for stability reasons, which is clearly unacceptable for high-throughput and rapid detection in the face of large-scale epidemic outbreaks.

Therefore, a novel time-of-flight mass spectrometry detection technology which can be used for clinically and rapidly identifying pathogenic microorganisms and is repetitive is needed for the requirements of large sample quantity, simple use method, less time consumption and good effect of clinically identifying pathogenic microorganisms.

Disclosure of Invention

As described above, in the prior art, it has been thought that if total microbial proteins are used as a sample to be subjected to mass spectrometry, wherein part of non-ribosomal proteins account for a part of mass spectrometry scores, the mass spectrometry evaluation scores are increased, and a considerable proportion of false positives occurs, thereby affecting the result determination. However, the inventors have surprisingly found that if a suitable combination of cell wall lysis reagent and mass spectrometry matrix is selected, and the lysis time is selected, mass spectrometry detection of total microbial protein can lead to a positive increase in a considerable part of the identification scores of the fluctuations in the judgment intervals III-IV and increase the unreliable results which originally need secondary identification to the reliable intervals without affecting the result of the judgment standard I, II, thereby avoiding the possibility of false negative.

Therefore, the principle of the invention is as follows: based on the existing commercial products and judgment standards, according to the different protein identification principles, the reagent composition and the kit for mass spectrum detection of the pretreatment of the microorganism sample are provided, wherein the reagent composition has the advantages of less steps, short time consumption and capability of efficiently extracting holoprotein of pathogenic bacteria to replace ribosomal protein, and experimental parameters and reagents are optimized. The kit can obtain a characteristic peak spectrogram with strong signals and high discrimination, and has more stable and reliable accuracy for final identification scores.

Based on the principle, the kit is improved from the existing reagent by the following steps: the component I is selected to be used for efficiently crushing cells and releasing total protein, and the component II is selected to be used for crushing cells and releasing total protein and is simultaneously used as a mass spectrum matrix to carry out pre-mass spectrum pretreatment. In particular, formic acid in component I is mainly used for cell disruption; acetonitrile in the component I and the component II can permeate into cells as an organic solvent, promote the release of polypeptide in the cells and effectively extract total protein.

Therefore, the first object of the present invention is to provide a reagent composition for the pretreatment of a time-of-flight mass spectrometry microorganism sample, which comprises the following main components: a component I: acetonitrile and formic acid; and (2) component II: acetonitrile, trifluoroacetic acid and alpha-cyano-4-hydroxycinnamic acid.

In one embodiment, wherein component I comprises:

50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, and the balance water.

The component II comprises:

52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, and the balance water.

In one embodiment, the initial concentration of acetonitrile is 99.7% by volume (Alfa. Co. Ltd.), the initial concentration of formic acid is 98% by volume (Sigma. Co. Ltd., NO: 94318-250 ml-F), the initial concentration of trifluoroacetic acid is 99% by volume or more (Sigma. Co. Ltd.), and the initial state of alpha-cyano-4-hydroxycinnamic acid is yellow powder (Sigma. Co. Ltd., NO: 70990-1G-F, prepared to be supersaturated in concentration and have yellow powder precipitated)

The second purpose of the invention is to provide the reagent composition for preparing the detection product for identifying the unknown microorganism.

In one embodiment, the product is a time-of-flight mass spectrometry detection kit for microorganisms comprising:

(1) reagent composition for pretreatment of microorganism sample

(2) Other mass spectrometry reagents;

in one embodiment, the kit may further comprise: negative quality control material, positive quality control material, sample application and target sheet for mass spectrum detection.

The third object of the present invention is a method for detecting microorganisms by the above reagent composition or detection product, which improves the peak pattern quality and improves the accuracy of identifying microorganisms by a time-of-flight mass spectrometry system, comprising the steps of:

(1) the microorganisms to be identified are isolated in a suitable medium to obtain single colonies.

(2) Using a sterile toothpick (or 1. mu.l inoculating loop), a portion of the appropriate single colony was picked up in a centrifuge tube containing 10. mu.l of component I, and cell disruption, protein and polypeptide release, and lysis was carried out for 5 minutes.

(3) Mu.l of the above solution was applied to the wells of the target plate and dried at room temperature.

(4) The microtube for component II was opened, and 1. mu.l of component II was removed and added to the same well site and dried at room temperature.

(5) And (4) placing the target plate into a time-of-flight mass spectrometry system for detection.

In one embodiment, the microorganism detection is the determination of the genus, species, subspecies or subtype of microorganism. In a particular embodiment, the detection of microorganisms is of a non-diagnostic purpose to detect pathogenic bacteria, contaminating bacteria, drug-resistant bacteria, and the like.

In another embodiment, the microorganism in any of the above embodiments is a microorganism in environmental pollution, a microorganism in food quarantine, a microorganism in import and export commodities, a drug-resistant microorganism in pharmaceutical research, or the like.

In one embodiment, the microorganism of any of the above embodiments is a prokaryotic microorganism, a eukaryotic microorganism. In a specific embodiment, the prokaryotic microorganism comprises a bacterium. The eukaryotic microorganism is fungi including yeast, mold, etc.

Technical effects

Trifluoroacetic acid in component II is also used for cell disruption, and can act synergistically with formic acid in component I to substantially disrupt cells and substantially release total proteins including ribosomal proteins.

The matrix alpha-cyano-4-hydroxycinnamic acid in the component II mainly has the functions of forming a cocrystallized film on the surface of a biomolecule, absorbing energy from laser by the matrix after laser irradiation generated by a flight time mass spectrum system, and transferring the energy to the biomolecule, so that the biomolecule is ionized. And (3) detecting and analyzing the ionized biomolecules by a time-of-flight mass spectrometry system.

The whole cell protein (including all proteins in the thallus and proteins of cell wall cell membranes) is extracted after the treatment of the kit, so that the characteristic peak intensity of the strain is effectively improved, and the identification accuracy of a time-of-flight mass spectrometer is improved.

The pretreatment of the sample is simple and convenient, and compared with the method of only treating and releasing ribosomal protein, the method has no obvious disadvantages of time and cost;

from the example results, it can be seen that, compared with the control, due to the control mass spectrometry matrix reagent, in the limited space for accommodating bacteria and short drying time, the matrix only partially breaks the cell wall and releases part of the ribosomal protein, and the cell wall protein (such as outer membrane protein, porin, lipoprotein) and enzyme protein in the cell matrix are not separated and released, so that only part of the ribosomal protein in the matrix is excited by the effective laser, thereby completing the mass spectrometry.

However, since the test microorganism is fully lysed in the presence of component I (10. mu.l of component I in a centrifuge tube for 5 minutes) in the present invention, the total intracellular protein is completely released. Therefore, a part of the sample is sucked in the matrix for detection, and the total protein in the matrix can be completely predicted to be excited by the effective laser, so that the mass spectrum is completed.

Therefore, the total protein is fully extracted by pretreating the microorganism, the detection effect is high in flux, the accuracy is stable, and the stability is unexpected compared with the stability of only detecting the ribosomal protein.

Principles and definitions

In the process of detecting substances by a mass spectrum of a time-of-flight mass spectrometry system, the substances to be detected are excited by laser in a vacuum environment under the assistance of a matrix and pass through a flight tube to a detector, and the time of different substances passing through the flight tube is in negative correlation with the molecular weights of the different substances, namely the higher the molecular weight is, the slower the flight speed is and the later the time of the different substances reaching the detector is. The detector can give a spectrum based on the time to reach it (which can be converted to molecular weight) and the intensity. Different microorganisms have different proteins, the whole proteins of the microorganisms are extracted after the microorganism samples are processed, different protein peak patterns, namely different microorganisms, containing different characteristic fingerprint spectrums are detected on a time-of-flight mass spectrometer, and different types of microorganisms can be distinguished by comparing the time-of-flight mass spectrometry system analysis software with the characteristic spectrogram of known pathogenic microorganism in a database, namely the identification result of the microorganism is given.

The term "matrix": the matrix alpha-cyano-4-hydroxycinnamic acid mainly has the functions of forming a cocrystallized film on the surface of a biomolecule, and after laser irradiation generated by a flight time mass spectrum system, the matrix absorbs energy from the laser and transfers the energy to the biomolecule, so that the biomolecule is ionized. And (3) detecting and analyzing the ionized biomolecules by a time-of-flight mass spectrometry system.

Drawings

FIG. 12B shows the appearance of a peak of Staphylococcus epidermidis treated by two methods (the red color of the upper image is CK-M direct smearing method, and the light green color of the lower image is the method and reagent of the invention)

FIG. 22B shows the difference in the appearance of peaks of Staphylococcus epidermidis treated by the two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 33B shows the appearance of peaks in Staphylococcus hominis treated by the two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 43B shows the difference in the appearance of peaks of Staphylococcus hominis treated by the two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 55B shows the appearance of peaks in enterococcus faecalis treated by two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 65B shows the difference in the appearance of peaks of enterococcus faecalis treated by the two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 77B shows the appearance of peaks in Streptococcus pneumoniae treated by two methods (the red color in the upper panel is CK-M direct smearing method, and the light green color in the lower panel is the method and reagent of the invention)

FIG. 87B shows the difference in the peak appearance of Streptococcus pneumoniae treated by the two methods (red in the upper panel is CK-M direct smearing method, while light green in the lower panel is the method and reagent of the invention)

FIG. 98B Peak appearance of Streptococcus agalactiae treated by the two methods (Red in the upper panel is CK-M direct smearing method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 108B shows the difference in the peak appearance of Streptococcus agalactiae treated by the two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 119B shows the appearance of a peak of E.coli treated by two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 129B shows the difference in the appearance of peaks of E.coli treated by the two methods (the red color of the upper panel is CK-M direct smearing method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 1310B shows the appearance of peaks after treatment of Enterobacter cloacae by two methods (the red color in the upper panel is CK-T acetonitrile extraction method, the light green color in the lower panel is the method and reagent of the invention)

FIG. 1410B shows the difference in the appearance of peaks of Enterobacter cloacae treated by the two methods (the red color in the upper panel is CK-T acetonitrile extraction method, and the light green color in the lower panel is the method and reagent of the present invention)

1511A Klebsiella pneumoniae produced by two methods (CK-T formic acid acetonitrile extraction method for red upper part, and the method and reagent for light green lower part)

FIG. 1611A shows the difference in peak appearance of Klebsiella pneumoniae treated by two methods (the red color in the upper panel is CK-T formic acid acetonitrile extraction method, and the light green color in the lower panel is the method and reagent of the present invention)

FIG. 1713B Peak appearance of Pseudomonas aeruginosa treated by two methods (CK-T formic acid acetonitrile extraction method for red upper panel, the method and reagent of the invention for light green lower panel)

FIG. 1813B the difference of peak appearance of Pseudomonas aeruginosa treated by two methods (the red color of the upper panel is CK-T acetonitrile formate extraction method, the light green color of the lower panel is the method and reagent of the present invention)

FIG. 1914A shows that Serratia marcescens treated by two methods (red in the upper part is CK-T acetonitrile formate extraction method, while light green in the lower part is the method and reagent of the present invention)

FIG. 2014A shows the difference of peak appearance of Serratia marcescens treated by two methods (the red color of the upper graph is CK-T formic acid acetonitrile extraction method, and the light green color of the lower graph is the method and the reagent of the invention)

FIG. 2117A is a graph showing the appearance of peaks of Aeromonas hydrophila treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 2217A shows the difference of the appearance of peaks of Aeromonas hydrophila treated by two methods (the red color of the upper panel is CK-T formic acid acetonitrile extraction method, the light green color of the lower panel is the method and reagent of the present invention)

FIG. 2338 shows the peak appearance of Klebsiella oxytoca treated by two methods (CK-T formic acid acetonitrile extraction method for red upper panel, the present invention for light green lower panel)

FIG. 2438 shows the difference in the peak-forming conditions of Klebsiella oxytoca treated by two methods (the red color in the upper panel is CK-T formic acid acetonitrile extraction method, and the light green color in the lower panel is the method and reagent of the present invention)

FIG. 25140 peak appearance of Streptococcus mutans treated by two methods (red color in upper panel is CK-T acetonitrile formate extraction method, while light green color in lower panel is the method and reagent of the invention)

26140 Peak Difference in Streptococcus mutans treated with the two methods (Red in the upper panel is CK-T acetonitrile extraction method, while light Green in the lower panel is the method and reagent of the invention)

FIG. 27159 Streptococcus mutans treated by the two methods showed a peak (red in the upper panel is CK-T acetonitrile formate extraction, while light green in the lower panel is the method and reagent of the invention)

FIG. 28159 differences in the appearance of peaks in Streptococcus mutans treated by the two methods (red in the upper panel is CK-T acetonitrile extracted from formic acid; light green in the lower panel is the method and reagent of the invention)

FIG. 29 SLT Lactobacillus rhamnosus treated with two methods and then the peak appearance (the red color of the upper panel is CK-T formic acid acetonitrile extraction method, the light green color of the lower panel is the method and reagent of the invention)

FIG. 30 shows the difference in the appearance of peaks of SLT Lactobacillus rhamnosus treated by two methods (the red color of the upper panel is CK-T formic acid acetonitrile extraction method, and the light green color of the lower panel is the method and reagent of the present invention)

FIG. 3130956 shows the appearance of peaks in Vibrio parahaemolyticus treated by two methods (the red color in the upper panel is CK-T acetonitrile formate extraction method, the light green color in the lower panel is the method and reagent of the present invention)

FIG. 3230956 shows the difference in the appearance of peaks of Vibrio parahaemolyticus treated by the two methods (the red color in the upper panel is CK-T acetonitrile formate extraction method, and the light green color in the lower panel is the method and reagent of the present invention)

FIG. 331A Peak appearance of Staphylococcus aureus treated by two methods (Red in the top panel is CK-M direct plating method, while light Green in the bottom panel is the method and reagent of the invention)

FIG. 341A shows the difference in the appearance of peaks in Staphylococcus aureus treated by the two methods (the red color in the upper panel is CK-M direct plating method, and the light green color in the lower panel is the method and reagent of the present invention)

FIG. 35E 35 peak appearance of Candida krusei treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 36E 35 shows the difference in the peak-off behavior of Candida krusei after post-treatment with two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 37E 39 peak appearance of Candida guilliermondii after treatment with two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, light green in the lower panel is the method and reagent of the present invention)

FIG. 38E 39 shows the difference in peak formation of Candida guilliermondii when treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 39E 53 Peak appearance of Candida rugosa treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 40E 53 peak difference of Candida rugosa treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 41E 55 peak appearance of Candida parapsilosis treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, and light green in the lower panel is the method and reagent of the present invention)

FIG. 42E 55 peak difference of Candida parapsilosis treated by two methods (red in the upper panel is CK-T formic acid acetonitrile extraction method, while light green in the lower panel is the method and reagent of the present invention)

FIG. 43E 58 peak appearance of Candida parapsilosis treated by two methods (CK-T acetonitrile extraction method in upper panel, method and reagent of the present invention in lower panel)

FIG. 44E 58 peak difference of Candida parapsilosis treated by two methods (the upper diagram is CK-T acetonitrile extraction method, the lower diagram is the method and reagent of the present invention)

Detailed Description

In order to further understand the technical features of the present invention, the present invention is described in detail with reference to the specific embodiments below. The embodiments are given by way of illustration only and not by way of limitation, and the scope of the invention should be determined by that of the claims which follow and that of insubstantial modifications made by those skilled in the art based on the teachings of the invention.

The first embodiment is as follows: the method of the invention and the method of directly coating the target plate (CK-M for short) in the traditional mass spectrometry are utilized to pretreat bacteria and then carry out mass spectrometry detection

Sampling a microorganism sample:

detailed information of the species, wherein the incubation conditions were all 5% C02 incubator, incubated for 24 h. As in table 1. The strains are all from the department of Hospital laboratory in Beijing coordination, and the generation time is 2015.11.2.

TABLE 1 detailed information of bacterial species (including medium, incubation time, culture conditions) in example one

Strain numbering	Experiment number	Chinese name of strain	Strain latin name	Culture medium	Pretreatment reagent and experimental method
						2B	2B-CK-M	Staphylococcus epidermidis	Staphylococcus epidermidis	BAP	Direct smearing method of CK
2B	2B-Y	Staphylococcus epidermidis	Staphylococcus epidermidis	BAP	Kits and methods of the invention
						3B	3B-CK-M	Human staphylococcus	Staphylococcus hominis	BAP	Direct smearing method of CK
3B	3B-Y	Human staphylococcus	Staphylococcus hominis	BAP	Kits and methods of the invention
						5B	5B-CK-M	Enterococcus faecalis	Enterococcus faecalis	BAP	Direct smearing method of CK
5B	5B-Y	Enterococcus faecalis	Enterococcus faecalis	BAP	Kits and methods of the invention
						7B	7B-CK-M	Streptococcus pneumoniae	Streptococcus penumoniae	BAP	Direct smearing method of CK
7B	7B-Y	Streptococcus pneumoniae	Streptococcus penumoniae	BAP	Kits and methods of the invention
						8B	8B-CK-M	Streptococcus agalactiae	Streptococcus agalactiae	BAP	Direct smearing method of CK
8B	8B-Y	Streptococcus agalactiae	Streptococcus agalactiae	BAP	Kits and methods of the invention
						9B	9B-CK-M	Escherichia coli	Escherichia coli	CBA/R	Direct smearing method of CK
9B	9B-Y	Escherichia coli	Escherichia coli	CBA/R	Kits and methods of the invention

(II) sample pretreatment and sample application:

the method of directly coating a target plate (CK-M) in the traditional mass spectrometry pretreatment method is adopted on a commercial TOF instrument, and mass spectrometry matrix reagents (50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 10mg/ml (M/v) alpha-cyano-4-hydroxycinnamic acid and the balance of water) of the CK-M are covered, so that the identification of the strain is carried out on the TOF instrument, and one strain is collected. The method comprises the following specific steps:

1) the microorganisms to be identified are isolated in a suitable medium to obtain single colonies.

2) Single colonies were picked with toothpicks and thinly spread on the target plate.

3) The mass spectrometry matrix reagent covered with 1ul CK-M was dried at room temperature.

4) And putting the target plate into a TOF instrument for mass spectrum detection.

5) And (5) putting the detected data into a biotyper of TOF for identification.

By using the pretreatment method (namely the microbial sample treatment reagent of the flight time mass spectrum system of Beijing-Yixinbo-Chuang biotechnology limited), detection is carried out on TOF, and a strain is collected at one point according to the identification and the scoring of a judgment interval I-IV. The method comprises the following specific steps:

2) Using a sterile toothpick (or 1. mu.l inoculating loop), a portion of the appropriate single colony was picked up in a centrifuge tube containing 10. mu.l of component I, and cell disruption, protein and polypeptide release, and lysis was carried out for 5 minutes.

3) Mu.l of the above solution was applied to the wells of the target plate and dried at room temperature.

4) The microtube for component II was opened, and 1. mu.l of component II was removed and added to the same well site and dried at room temperature.

5) And (4) placing the target plate into a time-of-flight mass spectrometry system for detection.

In this example, microbial samples were processed using different reagents and methods, and the target list schematic on a TOF instrument is shown in table 2:

the target plate of the TOF instrument was 16 × 24=384, and in the schematic diagram, the target was spotted after treatment by two methods according to the same species, respectively, and the treatment methods corresponding to the species numbers in the table (see table 1). Because the mass spectrometer detects proteins, peaks appear in places with proteins, and the difference between the types of total proteins (the invention) and ribosomal proteins (the traditional method) can be known by looking up peak diagrams (such as fig. 1-12) after the two methods are processed.

Table 2 schematic diagram of target list on TOF instrument in example one

1

2

3

4

5

6

…

24

A

2B-CK-M

2B-Y

3B-CK-M

3B-Y

5B-CK-M

5B-Y

B

…

C

D

E

…

P

(III) carrying out mass spectrum detection on Autoflex speed TOF/TOF and identifying on software biotyper

From the peak appearance (as shown in fig. 1-12), the peak appearance of the bacteria treated by the method and the reagent of the invention is more than that of the bacteria treated by the traditional method of directly coating the target plate, namely the peak of the non-ribosomal protein, so that the score is high (the score is shown in table 3). For example:

FIG. 2 shows that the number of the strain 2B has 4440.518Da and 6547.576Da more peaks than those of the strain produced by the traditional method; the score is improved from the interval (unreliable interval) of the traditional method with the non-identification result of less than 1.7 points IV to the possible genus of 1.8 points III, and the result of the method has identification significance.

FIG. 6 shows that the number of the strain 5B has 6231.249Da, 6677.828Da, 6863.325, 8887.616 and 9535.425Da more than the peak value of the conventional method; the score is increased from 1.99 to III possible genera to 2.134 to II reliable genera and possible species intervals. And a judgment standard interval is increased.

③ FIG. 12 shows that the number of the strain 9B has the peak values of 4188.513Da, 4767.497Da and 5148.902Da which are increased by using the method compared with the traditional method; the score is increased from the possible genus interval of 1.84 points IV to the species interval determined by 2.497 points I, which obviously improves the detection reliability and accuracy.

TABLE 3 identification of bacteria treated by the direct target coating method of the present invention and conventional methods

Example two: the method and the traditional mass spectrometry method are utilized to perform mass spectrometry detection after the extraction method of acetonitrile formate (CK-T for short) in the invention pretreats bacteria

Sampling a microorganism sample:

the detailed information of the species is shown in Table 4, wherein the culture conditions were the same as in example one. The strains are all from the department of Hospital laboratory in Beijing coordination, and the generation time is 2015.11.2.

TABLE 4 detailed information of bacterial species (including medium, incubation time, culture conditions) in example two

Strain numbering	Experiment number	Chinese name of strain	Strain latin name	Culture medium	Pretreatment reagent and experimental method
						10B	10B-CK-T	Enterobacter cloacae	Enterobacter cloacae	CBA/R	Method for extracting acetonitrile formate of CK
10B	10B-Y	Enterobacter cloacae	Enterobacter cloacae	CBA/R	Kits and methods of the invention
						11A	11A-CK-T	Klebsiella pneumoniae	klebsiella pneumonia	CBA/R	Method for extracting acetonitrile formate of CK
11A	11A-Y	Klebsiella pneumoniae	klebsiella pneumonia	CBA/R	Kits and methods of the invention
						13B	13B-CK-T	Pseudomonas aeruginosa	Pseudomonas aeruginosa	MH	Method for extracting acetonitrile formate of CK
13B	13B-Y	Pseudomonas aeruginosa	Pseudomonas aeruginosa	MH	Kits and methods of the invention
						14A	14A-CK-T	Serratia marcescens	Serratia marcescens	CBA/R	Method for extracting acetonitrile formate of CK
14A	14B-Y	Serratia marcescens	Serratia marcescens	CBA/R	Kits and methods of the invention
						17A	17A-CK-T	Hydrosophilic single package bacterium	Aeromonas hydrophila	CBA/R	Method for extracting acetonitrile formate of CK
17A	19A-Y	Hydrosophilic single package bacterium	Aeromonas hydrophila	CBA/R	Kits and methods of the invention
						38	38-CK-T	Acid-producing Klebsiella sp	klebsiella oxytoca	CBA/R	Method for extracting acetonitrile formate of CK
38	38-Y	Acid-producing Klebsiella sp	klebsiella oxytoca	CBA/R	Kits and methods of the invention

(II) sample pretreatment and sample application:

a commercial instrument Autoflex TOF/TOF is used, a formic acid acetonitrile extraction method (CK-T for short) in a traditional mass spectrum method pretreatment method is adopted, mass spectrum matrix reagents of the CK-T (50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 10mg/ml (m/v) alpha-cyano-4-hydroxycinnamic acid and the balance of water) are covered, strain identification is carried out on the TOF instrument, and one strain is collected. The method comprises the following specific steps:

1) placing a single colony in a 1.5ml Eppendorf tube, adding 300 mul of deionized water and 900 mul of absolute ethyl alcohol, fully and uniformly mixing, centrifuging at 13000 r/min for 2min, and removing supernatant;

2) centrifuging at 13000 r/min for 2min, and sucking and discarding the residual liquid;

3) adding 50 μ l of 70% formic acid, shaking, mixing, adding acetonitrile with equal amount, shaking, mixing, and centrifuging at 13000 r/min for 2 min;

4) dripping 1 μ l of the supernatant on a MALDI target plate, and drying at room temperature;

5) 1 mul of the matrix solution was applied to the sample spot and dried at room temperature;

6) and (6) performing detection on the machine.

By using the pretreatment method (namely the microbial sample treatment reagent of the flight time mass spectrum system of Beijing-Yixinbo-Chuang biotechnology limited), detection, identification and scoring are carried out on Autoflex speed TOF/TOF, and one strain is collected at one point. The method comprises the following specific steps:

In this example, microbial samples were processed using different reagents and methods, and the target list schematic on a TOF instrument is shown in table 5:

the target plate of the TOF instrument was 16 × 24=384, and in the schematic diagram, the target was spotted by two methods according to the same bacterial species, and the treatment methods corresponding to the bacterial species numbers in the table (see table 4). Because the mass spectrometer detects proteins, peaks appear in places with proteins, and the difference between the types of total proteins (the invention) and ribosomal proteins (the traditional method) can be known by looking up peak diagrams (such as fig. 13-24) after the two methods are processed.

Table 5 schematic diagram of target list on bruke's instrument in example two

1

2

3

4

5

6

…

24

A

10B-CK-T

10B-Y

11A-CK-T

11A-Y

13B-CK-T

13B-Y

B

…

C

D

E

…

P

(III) performing mass spectrum detection on TOF instrument and identifying on software biotyper

From the peak appearance (as shown in fig. 13-24), the peak appearance of the bacteria treated by the method and the reagent of the invention is more than that of the traditional acetonitrile formic acid extraction method, namely the peak of the non-ribosomal protein, so that the score is high (the score is shown in table 6). For example:

FIG. 14 shows that the strain number 10B has 2860.400Da, 3051.481Da, 3150.515Da, 3714.672Da, 3857.321Da, 4159.197Da, 4744.479Da, 4923.594Da more than the conventional method; the scoring is improved from the reliable genus possible species interval of 2.123 points II of the traditional method to the reliable species interval of 2.497 points I. And a judgment standard interval is increased.

FIG. 16 shows that the number of the strain with 11A has 8884.181Da and 9147.496Da peak values when the method is used compared with the traditional method; the score is increased from 1.989 to III possible genus interval to 2.225 to II reliable genus, possible species interval. And a judgment standard interval is increased.

③ FIG. 22 shows that the number of the strain with the number of 17A is 4713.251Da which is more than the peak value produced by the traditional method; the scoring is improved from 1.744 points IV possible genus interval to 2.032 points II determined species interval, which obviously improves the detection reliability and accuracy.

TABLE 6 identification of bacteria treated by acetonitrile formate extraction in the present invention and conventional methods

Example three: the method and the extraction method of acetonitrile formate (CK-T for short) in the traditional mass spectrometry method are utilized to treat oral pathogenic bacteria for mass spectrometry detection

Sampling of microorganism samples

3 specimens were taken from the North-university stomatology Hospital (the stored specimens were from patients in whom dental caries was diagnosed in the dental pulp department). The cells were inoculated into BHI medium for streaking. See table 7 for details, wherein the incubation conditions are all 5% C02 incubator for 18 h.

TABLE 7 oral pathogen sampling information

Strain numbering	Experiment number	Bacterial species name	Latin name of strain	Pretreatment reagent and experimental method
					159	159-M-T	Streptococcus mutans	streptococcus mutans	Method for extracting acetonitrile formate of CK
159	159-Y	Streptococcus mutans	streptococcus mutans	Kits and methods of the invention
					140	140-M-T	Streptococcus mutans	streptococcus mutans	Method for extracting acetonitrile formate of CK
140	140-Y	Streptococcus mutans	streptococcus mutans	Kits and methods of the invention
					741	741-M-T	Lactobacillus salivarius	lactobacillus salivarius	Method for extracting acetonitrile formate of CK
741	741-Y	Lactobacillus salivarius	lactobacillus salivarius	Kits and methods of the invention
					SLT	SLT-M-T	Lactobacillus rhamnosus	lactobacillus rhamnosus	Carboxylic acid acetonitrile of CKExtraction method
SLT	SLT-Y	Lactobacillus rhamnosus	lactobacillus rhamnosus	Kits and methods of the invention

(II) sample pretreatment and sample application:

6) and (6) performing detection on the machine.

By using the pretreatment method (namely the microbial sample treatment reagent of the flight time mass spectrum system of Beijing-Yixinbo-Biotech Co., Ltd.), identification and scoring of bacteria are carried out on a TOF instrument, and one strain is collected at one point. The method comprises the following specific steps:

In this example, microbial samples were processed using different reagents and methods, and the target list schematic on a TOF instrument is shown in table 8:

the target plate of the TOF instrument was 16 × 24=384, and in the schematic diagram, the target was spotted by two methods according to the same bacterial species, and the treatment methods corresponding to the bacterial species numbers in the table (see table 7). Because the mass spectrometer detects proteins, peaks appear in places with proteins, and the difference between the types of total proteins (the invention) and ribosomal proteins (the traditional method) can be known by looking up peak diagrams (such as fig. 25-fig. 30) after the two methods are processed.

Table 8 schematic diagram of target list on bruke's instrument in example three

1

2

3

4

5

6

…

24

A

159-CK-T

159-Y

140-CK-T

140-Y

741-CK-T

741-Y

B

…

C

D

E

…

P

(III) carrying out mass spectrum detection on a commercial instrument Autoflex speed TOF/TOF and identifying on a software biotype

The experimental strain spectrogram is shown in figures 25-30, and from the peak chart, the peak number of the bacteria treated by the method and the reagent is more than that of the bacteria treated by the traditional formic acid acetonitrile extraction method, namely the peak of the non-ribosomal protein, so that the score is high (the score is shown in table 9). For example:

in FIGS. 27 and 28, it can be seen that the strain number 159 has 5020.137Da peak value compared with the conventional method; the scoring is improved from 1.991 scoring of the possible genus interval of III to 2.011 scoring of the reliable genus and possible species interval of II in the traditional method. And a judgment standard interval is increased.

FIG. 29 and FIG. 30 show that the strain with the number SLT has 7209.869Da peak value compared with the conventional method; the scoring is improved from the reliable category of the possible species interval of 2.011 grade II to the reliable category interval of 2.34 grade I, a judgment standard interval is improved, and the reliability and the accuracy are obviously improved.

TABLE 9 details of the identification of oral pathogens

Example four: the method and the extraction method of acetonitrile formate (CK-T for short) in the traditional mass spectrometry method are utilized to carry out mass spectrometry detection on pathogenic bacteria-vibrio parahaemolyticus in food

(I) preparation of a microbiological sample

1) Sampling 25 g of scallop meat and body fluid of scallop to be detected by aseptic technique, adding 225mL of 3% sodium chloride alkali peptone water

Homogenizing with rotary blade type homogenizer at 8000 r/min for 1 min, or beating with beating type homogenizer for 2min to obtain product 1:10

Homogenizing the sample. If no homogenizer is available, putting the sample into a sterile mortar, taking a small amount of diluent from 225mL of 3% sodium chloride alkali peptone water, adding the diluent into the sterile mortar, grinding the sample, putting the ground sample into a 500 mL sterile conical flask, washing the residual sample in the mortar for 1-2 times by using the small amount of diluent, putting the washing liquid into the conical flask, finally putting all the residual diluent into the conical flask, and fully oscillating to prepare a 1:10 sample uniform liquid.

2) And culturing the sample homogeneous solution at 36 +/-1 ℃ for 8-18 h.

3) And (3) dipping a ring of enrichment fluid in a distance of 1cm below the liquid level by using an inoculating ring for all enrichment fluids which show growth, and scribing and separating on a TCBS (trichloromethane-butadiene-styrene) plate or a vibrio chromogenic culture medium plate. Culturing for 18-24 h at 36 +/-1 ℃.

4) A single colony was picked from the plate, numbered 30956.

5) The bacteria numbered 30956 were treated with the formic acid acetonitrile extraction method (abbreviated as CK-T) in the method of the present invention and the conventional mass spectrometry method, respectively. The strain information is shown in Table 10.

Information of strains cultured in shellfish meat and body fluid of Epimen 10 scallop

Strain numbering	Experiment number	Bacterial species name	Latin name of strain	Pretreatment reagent and experimental method
					30956	30956-CK-T	Vibrio parahaemolyticus	Vibrio Parahemolyticus	Method for extracting acetonitrile formate of CK
30956	30956-Y	Vibrio parahaemolyticus	Vibrio Parahemolyticus	Kit and method of the inventionMethod of producing a composite material

(II) sample pretreatment and sample application:

6) and (6) performing detection on the machine.

In this example, microbial samples were processed using different reagents and methods, and the target simple schematic on a TOF instrument is shown in table 11:

the target plate of the TOF instrument was 16 × 24=384, and in the schematic diagram, the target was spotted by two methods according to the same bacterial species, and the treatment methods corresponding to the bacterial species numbers in the table (see table 10). Because the mass spectrometer detects proteins, peaks appear in places with proteins, and the difference between the types of total proteins (the invention) and ribosomal proteins (the traditional method) can be known by checking peak graphs (such as figures 31-32) after the two methods are processed.

TABLE 11 example four TOF Instrument on target sheet schematic

	1	2	3	4	5	6	…	24
									A	30956-CK-T	30956-Y
B
									C
D
									E
…
									P

The experimental strain spectrogram is shown in fig. 31-32, and from the peak chart, the peak number of the bacteria treated by the method and the reagent is more than that of the bacteria treated by the traditional formic acid acetonitrile extraction method, namely the peak of the non-ribosomal protein, so that the score is high (the score is shown in table 12). For example:

in FIGS. 31 and 32, it can be seen that the strain number 30956 has a peak value of 5775.785Da when compared with the conventional method; the score is improved from the traditional method 1.995 score III possible genus interval to 2.007 score II reliable genus, possible species interval. Improves a judgment standard interval, reliability and accuracy

TABLE 12 identification of pathogens in food products treated by two methods

Example five: the method of the invention and the method of directly coating the target plate (CK-M for short) in the traditional mass spectrometry method are utilized to detect the staphylococcus aureus in the drainage ditch of the pigsty

(I) preparation of a microbiological sample

1) 25mL of the drainage ditch liquid was aspirated into 225mL of 7.5% NaCl broth or 10% NaCl trypticase soy broth in a sterile conical flask (a suitable number of sterile glass beads can be placed inside the flask) and shaken to mix.

2) And culturing the sample homogeneous solution at 36 +/-1 ℃ for 18-24 h.

3) And streaking the culture on a Columbia blood plate, and culturing for 18-24 h at 36 +/-1 ℃.

4) A single colony was picked from the plate and numbered 1A.

5) The bacteria numbered 1A are treated by the method of the invention and the method of extracting acetonitrile formate (CK-M for short) in the traditional mass spectrometry respectively. The strain information is shown in Table 13.

TABLE 13 Strain information in drainage ditches of hog houses

Strain numbering	Experiment number	Bacterial species name	Latin name of strain	Pretreatment reagent and experimental method
					1A	1A-CK-M	Staphylococcus aureus	Staphylococcus aureus	Direct smearing method of CK
1A	1A-Y	Staphylococcus aureus	Staphylococcus aureus	Kits and methods of the invention

(II) sample pretreatment and sample application:

a commercial instrument Autoflex TOF/TOF is used, a method (CK-M) of directly coating a target plate in a traditional mass spectrum method pretreatment method is adopted, mass spectrum matrix reagent {50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 10mg/ml (M/v) alpha-cyano-4-hydroxycinnamic acid and the balance of water } of the CK-M are covered, strain identification is carried out on the TOF instrument, and one strain is collected. The method comprises the following specific steps:

4) The target plate was placed in a bruke instrument for mass spectrometric detection.

5) And putting the detected data into a biotyper of the Bruk for identification.

In this example, microbial samples were processed using different reagents and methods, and the target list schematic on a TOF instrument is shown in table 14:

the target plate of the TOF instrument is a 16 × 24=384 target plate, and in the schematic diagram, the target is spotted after being treated by two methods according to the same strain, and the treatment methods corresponding to the numbers of the strains in the table are shown in table 13. Because the mass spectrometer detects proteins, peaks appear in places with proteins, and the difference between the types of total proteins (the invention) and ribosomal proteins (the traditional method) can be known by looking up peak graphs (fig. 33-34) after the two methods are processed.

TABLE 14 Bruk Instrument Upper target sheet schematic

	1	2	3	4	5	6	…	24
									A	1A-CK-M	1A-Y
B
									C
D
									E
…
									P

The experimental strain spectrogram is shown in fig. 33-34, and from the peak chart, the peak number of the bacteria treated by the method and the reagent is more than that of the bacteria treated by the traditional formic acid acetonitrile extraction method, namely the peak of the non-ribosomal protein, so that the score is high (the score is shown in table 15). For example:

in FIGS. 33 and 34, the number 1A of the strains has additional peak values of 2637.299Da, 2665.975Da, 3322.036Da, 3447.747Da, 3550.657Da, 4081.480Da, 4235.034Da, 4451.015Da and 4819.054Da compared with the conventional method; the score is improved from the traditional method 1.835 score III possible genus interval to 2.226 score II reliable genus, possible species interval. Improves a judgment standard interval, reliability and accuracy

TABLE 15 identification results of pathogens in drainage ditches of piggeries treated by two methods

Example six: the method and the traditional mass spectrometry method are utilized to perform mass spectrometry detection after the extraction method of acetonitrile formate (CK-T for short) in the invention pretreats fungi

Sampling a microorganism sample:

detailed information of the strains, wherein all the culture media are YPD culture media and all the conditions of the incubator are 37 ℃. As in table 16. The strain is from fungus laboratory of institute of microbiology of Chinese academy of sciences, and the generation time is 2015.11.2.

TABLE 16 example details of six fungal species (including medium, incubation time, culture conditions)

Strain numbering	Experiment number	Bacterial species name	Latin name of strain	Pretreatment reagent and experimental method
					E35	E35-CK-T	Candida Krusei	Candidia krusei	Method for extracting acetonitrile formate of CK
E35	E35-Y	Candida Krusei	Candidia krusei	Kits and methods of the invention
					E39	E39-CK-T	Candida guilliermondii	Candida guilliermondii	Method for extracting acetonitrile formate of CK
E39	E39-Y	Candida guilliermondii	Candida guilliermondii	Kits and methods of the invention
					E53	E53-CK-T	Candida rugosa	Candida rugosa	Method for extracting acetonitrile formate of CK
E53	E53-Y	Candida rugosa	Candida rugosa	Kits and methods of the invention
					E55	E55-CK-T	Candida parapsilosis	Candida pseudolambica	Method for extracting acetonitrile formate of CK
E55	E55-Y	Candida parapsilosis	Candida pseudolambica	Kits and methods of the invention
					E58	E58-CK-T	Candida parapsilosis	Candida pseudolambica	Method for extracting acetonitrile formate of CK
E58	E58-Y	Candida parapsilosis	Candida pseudolambica	Kits and methods of the invention

(II) sample pretreatment and sample application:

a method (CK-M) of directly coating a target plate in a traditional mass spectrum method pretreatment method is adopted on a commercial instrument Autoflex TOF/TOF, then mass spectrum matrix reagent {50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 10mg/ml (M/v) alpha-cyano-4-hydroxycinnamic acid and the balance of water } of the CK-M are covered, strain identification is carried out on the Autoflex TOF/TOF instrument, and one strain is collected. The method comprises the following specific steps:

6) and (6) performing detection on the machine.

The pretreatment method (namely, a microorganism sample treatment reagent of a flight time mass spectrum system of Beijing-Yixinbo-Chuang biotechnology limited) is used for detecting on an Autoflex speed TOF/TOF, and one strain is collected at one point according to the identification and scoring of a judgment interval I-IV. The method comprises the following specific steps:

In this example, microbial samples were processed using different reagents and methods, and the target simple diagrams on TOF instruments are shown in table 17:

the target plate of the TOF instrument was 16 × 24=384, and in the schematic diagram, the target was spotted by two methods for the same species, and the treatment methods corresponding to the species numbers in the table (see table 16). Because the mass spectrometer detects proteins, peaks appear in places with proteins, and the difference between the types of total proteins (the invention) and ribosomal proteins (the traditional method) can be known by looking up peak graphs (such as figures 35-42) after the two methods are processed.

TABLE 17 schematic diagram of target list on TOF instrument in example one

1

2

3

4

5

6

…

24

A

E35-CK-T

E35-Y

E39-CK-T

E39-Y

E53-CK-T

E53-Y

B

E55-CK-T

E55-Y

E58-CK-T

E58-Y

…

C

D

E

…

P

From the peak appearance (as shown in FIGS. 35-42), the bacteria treated by the method and the reagent of the present invention showed more peaks than the conventional acetonitrile formate method, i.e., the non-ribosomal protein peaks, and thus the score was high (as shown in Table 18). For example:

in FIGS. 35-36, the strain E35 has the peak values of 5906.755Da, 6278.609Da and 6942.736Da when compared with the conventional method; the scoring is improved from the 2.015-II determined possible species interval of the traditional method to the 2.39-I completely credible species interval, and the method results in that the detection reliability and accuracy are obviously improved.

From FIGS. 41 to 42, it can be seen that the strain with the number E58 has the peak values of 4325.176Da and 4637.467Da when compared with the conventional method, and the score is increased from 1.832 to III to 2.084 to II. The invention improves a judgment standard interval and has identification significance.

TABLE 18 identification of bacteria treated by acetonitrile formate extraction in the present invention and conventional methods

Claims

1. A reagent composition for time-of-flight mass spectrometry microbial sample pretreatment, comprising: a component I: acetonitrile and formic acid; and (2) component II: acetonitrile, trifluoroacetic acid and alpha-cyano-4-hydroxycinnamic acid.

2. The pretreatment reagent composition according to claim 1, wherein

The component I comprises: 50.0% (v/v) acetonitrile, 35.0% (v/v) formic acid, and the balance water;

the component II comprises: 52.5% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid, 15mg/ml (m/v) α -cyano-4-hydroxycinnamic acid, and the balance water.

3. A test product prepared from the reagent composition of claim 1 or 2 for identifying an unknown microorganism.

4. The test product of claim 3, wherein the test product is a time-of-flight mass spectrometry detection kit for microorganisms comprising:

(1) reagent composition for pretreatment of microorganism sample

(2) Other mass spectrometry reagents.

5. The test product of claim 4, wherein the kit further comprises: negative quality control material, positive quality control material, sample application and target sheet for mass spectrum detection.

6. The method for detecting the microorganisms by the reagent composition or the detection product improves the peak image quality and improves the accuracy of identifying the microorganisms by a time-of-flight mass spectrometry system, and comprises the following steps:

(1) separating the microorganism to be identified in a suitable culture medium to obtain a single colony;

(2) selecting a part of appropriate single colony by using a sterilized toothpick (or 1 mul inoculating loop) and placing the single colony in a centrifuge tube filled with 10 mul of the component I for cell disruption, releasing protein and polypeptide and cracking for 5 minutes;

(3) adding 1 μ l of the above solution to the hole of the target plate, and drying at room temperature;

(4) opening the microtube of the component II, transferring 1 mul of the component II to the same hole, and drying at room temperature;

7. The method of claim 6, wherein the microbial detection is determination of the genus, species, subspecies or subtype of microorganism.

8. The method of claim 6 or 7, wherein the microorganism is a microorganism in environmental pollution, a microorganism in food quarantine, a microorganism in import and export commodities, a drug-resistant microorganism in pharmaceutical research, or the like.

9. The method of claim 8, wherein the microorganism is a prokaryotic microorganism, a eukaryotic microorganism.

10. The method of claim 9, wherein the prokaryotic microorganism comprises a bacterium and the eukaryotic microorganism is a fungus comprising a yeast, a mold, and the like.