Multi-substrate-enzyme reaction-based portable small mass spectrum on-site rapid bacterial identification method

Abstract

The invention provides a method for portable mass spectrum identification of bacterial species based on substrate-enzyme reaction, which comprises the following steps: culturing to-be-detected bacteria to obtain a bacterial culture solution, washing and diluting to obtain a bacterial suspension; adding a plurality of pre-selected substrates into the bacterial suspension for co-incubation, so that the bacterial suspension and the pre-selected substrates are subjected to enzyme reaction to obtain a bacterial substrate mixed solution; detecting the bacterial substrate mixed solution by using electrospray ionization mass spectrometry to obtain the mass spectrum signal intensity of the preselected substrates and the enzyme reaction product so as to calculate the conversion degree data of each preselected substrate; in a coordinate system based on a preselected substrate type and a transformation degree, drawing a characteristic column map of the to-be-detected bacteria; and matching the characteristic column spectrum with the characteristic column spectrum in the bacterium variety database, and identifying the variety of the to-be-detected bacterium. The method disclosed by the invention can realize species identification of bacteria in the bacterial infection biological sample within 30 minutes, has the advantages of rapidness, accuracy and wide application range, and can be suitable for field detection of the bacterial sample.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a method for rapid on-site bacterial identification using a portable small-scale mass spectrometry system based on multi-substrate-enzyme reactions. Background Art

[0002] Globally, bacterial infections pose a serious threat to human health, and a large number of patients die from them every year. Therefore, rapid identification of pathogenic bacteria from food samples or body fluids is crucial for clinical treatment and disease prevention and control. At present, matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) technology has shown significant advantages in the field of clinical diagnosis. By comparing unknown bacterial samples with protein mass spectra in the database, combined with different microbial identification platforms, it has achieved a significant improvement in the accuracy of bacterial identification.

[0003] However, these technologies still have significant shortcomings. First, the identification process is still time-consuming and complex, relying on large mass spectrometers and professional operators. Second, the multi-step sample pretreatment process and the high requirements of large mass spectrometers for centralized laboratory environments, power supply and fast data connections limit their ability to be used for on-site diagnosis in resource-limited environments.

[0004] Therefore, in view of the urgent need for current bacterial detection technology, developing a rapid, portable and easy-to-operate bacterial species identification method to achieve rapid on-site detection of bacteria in biological samples has become a key issue that needs to be urgently addressed in the field of bacterial detection technology. Summary of the invention

[0005] In view of this, in order to at least partially solve at least one of the above-mentioned technical problems, the present disclosure provides a method for rapid on-site bacterial identification based on a portable small-scale mass spectrometry based on multi-substrate-enzyme reaction.

[0006] According to an embodiment of one aspect of the present disclosure, a method for identifying bacterial species by portable mass spectrometry based on substrate-enzyme reaction is provided, comprising:

[0007] Cultivating the bacteria extracted from the sample to be tested to obtain a bacterial culture solution of the sample to be tested, and washing and diluting the bacterial culture solution of the sample to be tested to obtain a bacterial suspension of the sample to be tested;

[0008] Adding a plurality of preselected substrates to the bacterial suspension of the sample to be tested for co-incubation, so that the bacterial suspension of the sample to be tested and the preselected substrates undergo an enzyme reaction to obtain a bacterial substrate mixed solution containing an enzyme reaction product;

[0009] The bacterial substrate mixture is detected by electrospray mass spectrometry to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product, so as to calculate the conversion degree data for each preselected substrate;

[0010] According to the conversion degree data of a plurality of preselected substrates, a characteristic column map of bacteria in the sample to be tested is drawn in a coordinate system based on the preselected substrate types and conversion degrees;

[0011] The characteristic column map of bacteria in the sample to be tested is matched with the characteristic column map in the bacterial species database to identify the species of bacteria in the sample to be tested.

[0012] According to an embodiment of the present disclosure, a method for rapid on-site bacterial identification based on a portable small-scale mass spectrometry of multi-substrate-enzyme reaction is provided for rapid identification of bacterial species. The bacteria react with preselected substrates, and specific enzymes in the bacteria catalyze the conversion of substrates into detectable products. The mass spectrometry signal of the preselected substrate and its product is detected by mass spectrometry analysis technology to obtain data on the degree of substrate conversion, and based on the data on the degree of substrate conversion, a characteristic bar graph of the bacteria is drawn to obtain data characteristics of the unique degree of conversion produced after the bacteria react with the substrate. By matching and comparing the characteristic bar graph of unknown bacteria in the sample to be tested with the characteristic bar graph in the bacterial species database, rapid identification of bacterial species is achieved. The method for identification of bacteria in the present disclosure can be combined with portable small-scale mass spectrometry detection, and can achieve identification of bacterial species in bacterial infection biological samples within 30 minutes. It has the advantages of being fast, accurate, and having a wide range of applications. It can be flexibly applied to on-site detection of various bacterial samples and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the process of identifying bacterial species disclosed in the present invention;

[0014] Figure 2 The mass spectra of the preselected substrate and product in Example 1 of the present disclosure;

[0015] Figure 3 is a characteristic column map of bacteria in Example 2 of the present disclosure;

[0016] Figure 4 is a bacterial species typing diagram of the bacteria in Example 2 of the present disclosure;

[0017] Figure 5 is a characteristic column map of bacteria in Example 3 of the present disclosure;

[0018] Figure 6 is a bacterial species typing diagram of the bacteria in Example 3 of the present disclosure;

[0019] Figure 7is a characteristic column map of bacteria in Example 4 of the present disclosure;

[0020] Figure 8 This is a bacterial species classification diagram of the bacteria in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in conjunction with specific embodiments.

[0022] The endpoints and any values ​​of the ranges disclosed in this disclosure are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this disclosure.

[0023] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. The terms "comprise", "include", etc. used herein indicate the existence of the features, steps, operations and / or components, but do not exclude the existence or addition of one or more other features, steps, operations or components.

[0024] Faced with the serious threat posed by bacterial infections to human health worldwide, rapid and accurate bacterial detection technology is essential to improving the level of public health. Rapid and accurate diagnosis of infectious bacteria enables clinicians to quickly target patients for treatment, and early diagnosis of bacteria can reduce the morbidity and mortality of patients. However, currently commonly used technologies such as MALDI-TOF MS have problems such as time-consuming identification process, complex operation, and reliance on large mass spectrometers and professional operators. In addition, the multi-step sample pretreatment process and the high requirements of large mass spectrometers for centralized laboratory environment, power supply, etc. limit their on-site instant identification capabilities in resource-limited environments. Therefore, there is an urgent need to develop a rapid, portable and easy-to-operate method for bacterial species identification.

[0025] In the process of implementing the present disclosure, it is found that bacteria produce specific enzymes during growth and metabolism, and these enzymes can catalyze specific chemical reactions of substrates to generate detectable products with bacterial specificity. In this regard, the present disclosure proposes a method for rapid on-site bacterial identification based on a portable small mass spectrometer with multi-substrate-enzyme reaction, which is used to quickly identify bacterial species. The present disclosure applies substrate-enzyme reaction to the identification process of bacterial species, and by selecting a preselected substrate with bacterial-specific enzymatic reaction, the bacteria react with the preselected substrate to generate a product with bacterial specificity. And the mass spectrometry technology is used to detect the mass spectrometry signal intensity of the substrate and the product to calculate the specific data of the substrate conversion degree, which can directly reflect the activity of the bacterial-specific enzymatic reaction. Further, based on the substrate conversion degree data, a characteristic columnar spectrum is drawn and matched with the characteristic columnar spectrum in the bacterial species database, so as to realize the rapid and accurate identification of the bacterial species to be tested. The entire detection process does not require complicated pre-treatment steps, and the operation is simple and fast. A small portable mass spectrometer can be used to complete the identification of bacterial species in bacterial infection biological samples within 30 minutes at the fastest. It not only improves detection efficiency, but also broadens application scenarios. It is particularly suitable for on-site testing, disease monitoring, and public health emergency response. It provides strong technical support for improving public health levels, guiding clinicians' targeted treatments, and reducing patients' morbidity and mortality.

[0026] Figure 1 Schematic diagram of the process for identifying bacterial species disclosed in the present invention.

[0027] like Figure 1 As shown, according to an embodiment of one aspect of the present disclosure, a method for identifying bacterial species by portable mass spectrometry based on substrate-enzyme reaction is provided, comprising:

[0028] Cultivating the bacteria extracted from the sample to be tested to obtain a bacterial culture solution of the sample to be tested, and washing and diluting the bacterial culture solution of the sample to be tested to obtain a bacterial suspension of the sample to be tested;

[0029] Adding a plurality of preselected substrates to the bacterial suspension of the sample to be tested for co-incubation, so that the bacterial suspension of the sample to be tested and the preselected substrates undergo an enzyme reaction to obtain a bacterial substrate mixed solution containing an enzyme reaction product;

[0030] The bacterial substrate mixture is detected by electrospray mass spectrometry to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product, so as to calculate the conversion degree data for each preselected substrate;

[0031] According to the conversion degree data of a plurality of preselected substrates, a characteristic column map of bacteria in the sample to be tested is drawn in a coordinate system based on the preselected substrate types and conversion degrees;

[0032] The characteristic column map of bacteria in the sample to be tested is matched with the characteristic column map in the bacterial species database to identify the species of bacteria in the sample to be tested.

[0033] According to an embodiment of the present disclosure, according to an embodiment of the present disclosure, a portable small-scale mass spectrometry method for rapid on-site bacterial identification based on multi-substrate-enzyme reaction is provided, which is used to quickly identify bacterial species. The bacteria react with preselected substrates, and specific enzymes in the bacteria catalyze the substrates into detectable products. The mass spectrometry signals of the preselected substrates and their products are detected using mass spectrometry analysis technology to obtain data on the degree of substrate conversion. Based on the substrate conversion degree data, a characteristic bar graph of the bacteria is drawn to obtain data characteristics of the unique degree of conversion produced after the bacteria react with the substrates. By matching and comparing the characteristic bar graph of unknown bacteria in the sample to be tested with the characteristic bar graph in the bacterial species database, rapid identification of bacterial species can be achieved.

[0034] According to an embodiment of the present disclosure, a method for establishing a bacterial species database includes:

[0035] For different pure strains of known bacteria, culture, co-incubation, mass spectrometry detection and mapping operations are performed to obtain characteristic column maps of different pure strains of known bacteria, forming a bacterial species database. The bacterial species database stores the unique conversion degree data characteristics produced after different types of bacteria react with pre-selected substrates, providing an important basis for the identification of unknown bacterial species. Among them, known pure strains of bacteria include Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and any one or more common infectious bacterial species in Enterobacteriaceae.

[0036] According to an embodiment of the present disclosure, the method for establishing a bacterial species database specifically includes:

[0037] Cultivate pure bacteria of known species, wash and dilute to obtain a bacterial suspension;

[0038] Adding a reaction reagent comprising a plurality of preselected substrates to a bacterial suspension for co-incubation, wherein at least one of the plurality of preselected substrates is capable of undergoing an enzyme reaction with pure bacteria to form a bacterial substrate mixed solution comprising an enzyme reaction product;

[0039] Using electrospray mass spectrometry to perform mass spectrometry detection on the bacterial substrate mixture to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product, so as to calculate the conversion degree data for each preselected substrate;

[0040] According to the conversion degree data of multiple pre-selected substrates, a characteristic bar graph of pure bacteria is drawn in a coordinate system based on the pre-selected substrate types and conversion degrees;

[0041] For different pure strains of known bacteria, the operations of culturing, co-incubation, mass spectrometry detection and mapping are repeated to obtain characteristic column maps of different pure strains of known bacteria, forming a bacterial species database. In the above steps, the specific operations of culturing, washing, dilution, co-incubation, mass spectrometry detection and mapping are similar to the method for identifying bacterial species disclosed in the present invention, and will not be repeated here.

[0042] According to an embodiment of the present disclosure, the method for identifying bacterial species further includes: when the bacterial species extracted from the sample to be tested is not included in the bacterial species database, combining other bacterial species identification methods, such as bacterial gene sequencing detection, MALDI-MS mass spectrometry detection, bacterial phenotypic testing, etc., after determining the bacterial species, the characteristic bar graph of the bacteria extracted from the sample to be tested is added to the bacterial species database, and by updating and expanding the database, it includes characteristic graphs of more bacterial species, thereby providing support for rapid detection of the same type of bacteria in the future.

[0043] According to the disclosed embodiment, the preselected substrate includes any two or more of L-glutamyl-3-carboxyl-4-nitroaniline, L-proline aniline, 4-aminophenyl-β-D-galactoside, L-tyrosine-4-nitroaniline, L-pyroglutamic acid-2-naphthylamide, phenyl-2-acetylamino-2-deoxy-α-D-pyranoglucoside, and L-alanine-4-nitroanilide, preferably 2 to 5. The preselected substrate can be a variety of combinations of any substrates to cover a wider range of bacterial metabolic characteristics, thereby improving the accuracy and comprehensiveness of identification. The preselected substrate concentration is 0.1 to 10 ppm, for example, 0.1 ppm, 1 ppm, 3 ppm, 5ppm, 7 ppm, 10 ppm, etc., and the dosage is 0.5 to 40 μL, for example, 0.5 μL, 1 μL, 10 μL, 20 μL, 30 μL, 40 μL, etc. Ensure that the bacteria have enough substrate to metabolize in the reaction, while avoiding reaction inhibition caused by excessive substrate. In addition, the concentration and dosage of the substrate further affect the signal intensity of mass spectrometry detection and the accuracy of identification results.

[0044] According to the embodiments of the present disclosure, the culture medium includes any one of agar medium, nutrient broth medium, and LB liquid medium, which can meet the nutritional requirements of different bacteria; the culture time is 8 to 16 hours, for example, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, etc. The appropriate culture time period allows the bacteria to have enough time to grow and reproduce, while avoiding the depletion of the culture medium or autolysis of the bacteria that may be caused by excessive growth; the culture temperature is 25°C to 40°C, for example, 25°C, 30°C, 35°C, 40°C, etc. The temperature range of 25°C to 40°C is suitable for the growth of most bacteria and can ensure their normal physiological metabolic activities. The concentration of the bacterial suspension is 10 7~10 9 cells / mL, for example, 10 7 cells / mL, 10 8 cells / mL, 10 9 cells / mL, etc., to ensure that there are sufficient bacteria in subsequent tests to obtain reliable test results, while avoiding insufficient nutrition of the culture medium due to excessive bacteria.

[0045] According to the embodiment of the present disclosure, in specific operation, the process of culturing, washing and diluting bacteria includes:

[0046] Under sterile conditions, inoculate pure bacteria of known species into appropriate culture medium and culture overnight in a shaker at 37°C and 220 rpm;

[0047] After the culture is completed, the culture medium containing bacteria is transferred to a centrifuge tube for washing, and centrifuged at 1000 rpm to 12000 rpm for 3 min to 5 min, and then the upper culture medium supernatant is discarded, and the bacterial precipitate is retained;

[0048] The collected bacterial pellet was resuspended in 1 mL of deionized water and the bacteria were evenly dispersed by pipetting;

[0049] Repeat the washing and resuspension steps 2 to 3 times and dilute with deionized water to OD 600 =0.9, forming a uniform bacterial suspension.

[0050] According to the embodiments of the present disclosure, the co-incubation temperature is 25°C~45°C, for example, it can be 25°C, 30°C, 35°C, 40°C, 45°C, etc., to ensure that the reaction between the bacteria and the substrate can be carried out under suitable conditions; the co-incubation time is 5min~240 min, for example, it can be 5 min, 10 min, 30 min, 60 min, 120 min, 240 min, etc., preferably 5min~30 min. The bacterial identification method of the present disclosure does not require complicated processing and operation when used, and the co-incubation time can be shortened to within 30 minutes, effectively improving the detection efficiency; the rotation speed during the co-incubation process is 200~300rpm, for example, it can be 200rpm, 220rpm, 250rpm, 270rpm, 300rpm, etc., to ensure that the bacteria are fully mixed with the substrate during the incubation process, and promote the rapid and uniform bacteria-enzyme reaction.

[0051] According to an embodiment of the present disclosure, mass spectrometry detection of a bacterial substrate mixture using electrospray mass spectrometry includes:

[0052] The bacterial substrate mixed solution is injected into the capillary and the electrospray solvent is added. A voltage is applied to the capillary to generate an ionized spray. The ionized spray is detected by mass spectrometry to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product.

[0053] According to the embodiments of the present disclosure, a portable small-scale mass spectrometry detection instrument using electrospray mass spectrometry can be used in specific operations, so that mass spectrometry detection can be performed on-site or in an environment with limited resources, which greatly expands the scope of use of mass spectrometry technology, making it not only limited to traditional laboratory environments, but also able to cope with diverse needs such as emergencies, on-site detection or field work.

[0054] According to an embodiment of the present disclosure, the ion intensity of the substrate in the mass spectrum is expressed as I 底物 The ion intensity of the corresponding product in the mass spectrum is represented by I 产物 The conversion degree of the substrate is calculated by the following formula:

[0055] Degree of conversion of substrate = I 底物 / (I 底物 +I 产物 )

[0056] When the conversion degree of the substrate is greater than 0, it indicates that a substrate-enzyme reaction has occurred between the substrate and the enzyme in the bacteria, indicating the presence of a specific bacterial species.

[0057] According to the disclosed embodiment, the capillary diameter is 1 to 10 μm, which can be prepared using a P1000 needle puller, and the capillary diameter can be, for example, 1 μm, 3 μm, 5 μm, 7 μm, 10 μm, etc. A smaller capillary diameter helps to produce smaller droplets, which can increase the surface area of ​​the sample, thereby improving the ionization efficiency and the intensity of the mass spectrometry signal. The applied voltage is a DC voltage or an AC voltage, and the voltage is 1.5 to 5 kV, for example, 1.5 kV, 2 kV, 3 kV, 4 kV, 5 kV, etc. The voltage can be adjusted according to the specific bacterial substrate mixture. The spray solvent is an organic solvent, including at least one of methanol, acetonitrile, and acetone. Selecting an organic solvent with good volatility and conductivity helps the dissociation and ionization of the bacterial substrate mixture, while reducing the residue after mass spectrometry analysis.

[0058] According to an embodiment of the present disclosure, the above identification method also includes: reducing the dimension and visualizing the conversion degree data of the substrate to classify different types of bacteria.

[0059] According to the embodiments of the present disclosure, a large amount of high-dimensional substrate conversion degree data can be analyzed and reduced in dimension by using a machine learning algorithm for dimensionality reduction, such as the tSNE method, so that the complex substrate conversion degree data can be converted into a visual form, and different types of bacteria can be effectively typed.

[0060] According to an embodiment of the present disclosure, the sample to be tested includes at least one of whole blood, cell-infected tissue, and urine, and the identification method of the present disclosure is suitable for identification of bacterial species in different application scenarios.

[0061] In order to make the purpose, technical scheme and advantages of the present disclosure more clear, the present disclosure is further described in detail below in conjunction with specific embodiments. Specific techniques or conditions not specified in the embodiments are conventional methods and can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0062] Embodiment 1:

[0063] Example 1 of the present disclosure identifies the species of five common clinical bacteria, including the following steps:

[0064] Under sterile conditions, five common clinical bacteria: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterococcus faecium were inoculated into 2 mL of LB medium and cultured overnight in a shaker at 37°C and 220 rpm.

[0065] After the culture is completed, the culture medium containing bacteria is transferred to a centrifuge tube for washing and centrifuged at 3000 rpm for 3 minutes. The supernatant of the culture medium is then discarded and the bacterial precipitate is retained. The collected bacterial precipitate is resuspended in 1 mL of deionized water and the bacteria are evenly dispersed by pipetting. After repeating the washing and resuspending steps twice, the precipitate is diluted with deionized water to an OD of 0. 600 = 0.9, forming a concentration of 10 9 cells / mL of bacterial suspension.

[0066] Four preselected substrates with a concentration of 100 ppm and 1 μL were added to the five bacterial suspensions respectively: L-proline aniline (substrate 1), 4-aminophenyl-β-D-galactoside (substrate 2), L-tyrosine-4-nitroaniline (substrate 3), and L-pyroglutamic acid-2-naphthamide (substrate 4). The bacterial suspensions and preselected substrates were placed in a constant temperature incubation device and incubated at 37°C and 200 rpm for 1 hour to form five bacterial substrate mixtures containing enzyme reaction products.

[0067] A capillary with a tip diameter of 5 μm was pulled using a P1000 needle puller, and the bacterial substrate mixture was extracted using a syringe and injected into the capillary.

[0068] Electrospray mass spectrometry was used to detect five bacterial substrate mixtures. The capillary was placed at the front end of the small mass spectrometer inlet and a voltage of 2 kV was applied to obtain the mass spectrometry signal intensity of the preselected substrate and enzyme reaction products. Figure 2The mass spectra of the preselected substrates and products in Example 1 of the present disclosure.

[0069] like Figure 2 As shown, the signal intensities of 4 preselected substrates and 4 products in the mass spectrum can be observed. According to the ion intensity of the preselected substrates in the mass spectrum and the ion intensity of the products in the mass spectrum, the conversion degree data for each preselected substrate is calculated.

[0070] According to the conversion degree data of multiple preselected substrates, in a coordinate system based on the preselected substrate types and conversion degrees, with the conversion degree of the substrate as the ordinate and the type of the preselected substrate as the abscissa, characteristic column graphs of the five bacteria were drawn respectively.

[0071] The characteristic column maps of the five bacteria were matched with the characteristic column maps in the bacterial species database to identify the species of the five bacteria.

[0072] Furthermore, with the help of mathematical statistics methods, the conversion degree data of the substrates calculated above were reduced in dimension and visualized for analysis, and the five types of bacteria were classified.

[0073] Example 2

[0074] Example 2 of the present disclosure identifies the bacterial species in whole blood, including the following steps:

[0075] The strains in the whole blood were cultured according to the standard specifications of the Clinical and Laboratory Standards Institute (CLSI) of the United States, inoculated into sheep blood agar medium, and streaked at 37°C for 24 hours. The streaking was repeated 2 to 3 times to obtain single colonies of bacteria in the whole blood.

[0076] Under sterile conditions, a single colony was inoculated into 2 mL of LB medium and cultured overnight in a shaker at 37° C. and 220 rpm.

[0077] After the culture is completed, the culture medium containing bacteria is transferred to a centrifuge tube for washing and centrifuged at 3000 rpm for 3 minutes. The supernatant of the culture medium is then discarded and the bacterial precipitate is retained. The collected bacterial precipitate is resuspended in 1 mL of deionized water and the bacteria are evenly dispersed by pipetting. After repeating the washing and resuspending steps twice, the precipitate is diluted with deionized water to an OD of 0. 600 = 0.9, forming a concentration of 10 9 cells / mL of bacterial suspension.

[0078] Four pre-selected substrates were added to the bacterial suspension at a concentration of 100 ppm and 1 μL respectively: L-glutamyl-3-carboxy-4-nitroaniline (substrate 1), L-proline aniline (substrate 2), 4-aminophenyl-β-D-galactoside (substrate 3), L-tyrosine-4-nitroaniline (substrate 4), and the bacterial suspension and the pre-selected substrates were placed in a constant temperature incubation device and incubated at 37°C and 200 rpm for 1 hour to form a bacterial substrate mixture containing enzyme reaction products.

[0079] A capillary with a tip diameter of 5 μm was pulled using a P1000 needle puller, and the bacterial substrate mixture was extracted using a syringe and injected into the capillary.

[0080] Electrospray mass spectrometry was used to detect the bacterial substrate mixture. The capillary was placed at the front end of the small mass spectrometer inlet and a voltage of 2kV was applied to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product. The conversion degree data for each preselected substrate was calculated based on the ion intensity of the preselected substrate in the mass spectrometer and the ion intensity of the product in the mass spectrometer.

[0081] According to the conversion degree data of multiple preselected substrates, in a coordinate system based on the preselected substrate types and conversion degrees, characteristic column graphs of bacteria are drawn respectively with the conversion degree of the substrate as the ordinate and the type of the preselected substrate as the abscissa. Figure 3 This is a characteristic bar graph of the bacteria in Example 2 of the present disclosure.

[0082] like Figure 3 As shown, it can be observed that there are obvious differences in the characteristic columnar spectra of the two bacteria in Example 2 of the present disclosure. The characteristic columnar spectra of the bacteria are matched with the characteristic columnar spectra in the bacterial species database to identify the species of the bacteria. The bacterial species in the whole blood can be identified as Escherichia coli and Klebsiella pneumoniae.

[0083] Furthermore, with the help of mathematical statistics methods, the conversion degree data of the substrate obtained by the above calculations were reduced in dimension and visualized for bacteria classification. Figure 4 This is a bacterial species classification diagram of the bacteria in Example 2 of the present disclosure.

[0084] like Figure 4 As shown, through dimensionality reduction analysis technology, high-dimensional data is converted into low-dimensional data, while retaining important difference information, so as to achieve bacterial typing and distinguish two types of bacteria, indicating that bacteria can be distinguished and identified through the characteristic bar graph based on conversion degree data provided by the present invention.

[0085] Example 3

[0086] Example 3 of the present disclosure identifies the bacterial species in urine, including the following steps:

[0087] Under sterile conditions, the bacteria extracted from urine were inoculated into 2 mL of LB medium and cultured overnight in a shaker at 37°C and 220 rpm.

[0088] After the culture is completed, the culture medium containing bacteria is transferred to a centrifuge tube for washing and centrifuged at 3000 rpm for 3 minutes. The supernatant of the culture medium is then discarded and the bacterial precipitate is retained. The collected bacterial precipitate is resuspended in 1 mL of deionized water and the bacteria are evenly dispersed by pipetting. After repeating the washing and resuspending steps twice, the precipitate is diluted with deionized water to an OD of 0. 600 = 0.9, forming a concentration of 10 9 cells / mL of bacterial suspension.

[0089] Four preselected substrates at a concentration of 100 ppm and 1 μL were added to the five bacterial suspensions: (4-aminophenyl-β-D-galactoside (substrate 1), L-tyrosine-4-nitroanilide (substrate 2), L-alanine-4-nitroanilide (substrate 3), L-pyroglutamic acid-2-naphthamide (substrate 4)), the bacterial suspension and the preselected substrates were placed in a constant temperature incubation device, and incubated at 37°C and 200 rpm for 1 hour to form a bacterial substrate mixture containing enzyme reaction products.

[0090] A capillary with a tip diameter of 5 μm was pulled using a P1000 needle puller, and the bacterial substrate mixture was extracted using a syringe and injected into the capillary.

[0091] Electrospray mass spectrometry was used to detect the bacterial substrate mixture. The capillary was placed at the front end of the small mass spectrometer inlet and a voltage of 2kV was applied to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product. The conversion degree data for each preselected substrate was calculated based on the ion intensity of the preselected substrate in the mass spectrometer and the ion intensity of the product in the mass spectrometer.

[0092] According to the conversion degree data of multiple preselected substrates, in a coordinate system based on the preselected substrate types and conversion degrees, characteristic column graphs of bacteria are drawn respectively with the conversion degree of the substrate as the ordinate and the type of the preselected substrate as the abscissa. Figure 5 This is a characteristic bar graph of the bacteria in Example 3 of the present disclosure.

[0093] like Figure 5 As shown, it can be observed that there are obvious differences in the characteristic histograms of the five bacteria in Example 3 of the present disclosure. The characteristic histogram of the bacteria is matched with the characteristic histogram in the bacterial species database to identify the species of the bacteria. The bacterial species in urine that can be identified are Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterobacter faecalis.

[0094] Furthermore, with the help of mathematical statistics methods, the conversion degree data of the substrate obtained by the above calculations were reduced in dimension and visualized for bacteria classification. Figure 6 This is a bacterial species classification diagram of the bacteria in Example 3 of the present disclosure.

[0095] like Figure 6 As shown, by using dimensionality reduction analysis technology, high-dimensional data is converted into low-dimensional data, while retaining important difference information, achieving bacterial typing and distinguishing 5 types of bacteria. It shows that the characteristic bar graph based on the conversion degree data provided by the present disclosure can distinguish and identify the 5 types of bacteria.

[0096] Example 4

[0097] Example 4 of the present disclosure identifies the types of bacteria in bacterially infected tissues, including the following steps:

[0098] The infected tissue was wiped with sterile cotton, and the sterile cotton after wiping the infected tissue was put into 1 mL of deionized water to elute the bacteria to obtain an eluate containing bacteria.

[0099] The strain was cultured according to the standard specifications of CLSI, inoculated into sheep blood agar medium, and streaked at 37°C for 24 h. The streaking was repeated 2 to 3 times to obtain single colonies of bacteria in the infected tissue.

[0100] Under sterile conditions, the obtained single colony was inoculated into 2 mL of LB medium and cultured overnight in a shaker at 37°C and 220 rpm;

[0101] After the culture is completed, the culture medium containing bacteria is transferred to a centrifuge tube for washing and centrifuged at 3000 rpm for 3 minutes. The supernatant of the culture medium is then discarded and the bacterial precipitate is retained. The collected bacterial precipitate is resuspended in 1 mL of deionized water and the bacteria are evenly dispersed by pipetting. After repeating the washing and resuspending steps twice, the precipitate is diluted with deionized water to an OD of 0. 600 = 0.9, forming a concentration of 10 9 cells / mL of bacterial suspension.

[0102] Four pre-selected substrates were added to the bacterial suspension at a concentration of 100 ppm and 1 μL respectively: L-tyrosine-4-nitroanilide (substrate 1), L-pyroglutamic acid-2-naphthylamide (substrate 2), phenyl-2-acetylamino-2-deoxy-α-D-pyranoglucopyranoside (substrate 3), and L-alanine-4-nitroanilide (substrate 4). The bacterial suspension and the pre-selected substrates were placed in a constant temperature incubation device and incubated at 37°C and 200 rpm for 1 hour to form a bacterial substrate mixture containing enzyme reaction products.

[0103] Electrospray mass spectrometry was used to detect the bacterial substrate mixture. The capillary was placed at the front end of the small mass spectrometer inlet and a voltage of 2kV was applied to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product. The conversion degree data for each preselected substrate was calculated based on the ion intensity of the preselected substrate in the mass spectrometer and the ion intensity of the product in the mass spectrometer.

[0104] According to the conversion degree data of multiple preselected substrates, in a coordinate system based on the preselected substrate types and conversion degrees, characteristic column graphs of bacteria are drawn respectively with the conversion degree of the substrate as the ordinate and the type of the preselected substrate as the abscissa. Figure 7 This is a characteristic bar graph of the bacteria in Example 4 of the present disclosure.

[0105] like Figure 7 As shown, it can be observed that there are obvious differences in the characteristic bar graphs of the three bacteria in Example 4 of the present disclosure.

[0106] The characteristic column map of the bacteria is matched with the characteristic column map in the bacterial species database to identify the species of the bacteria. The bacterial species in the bacterial infection tissue can be identified as Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacter faecalis.

[0107] Furthermore, with the help of mathematical statistics methods, the conversion degree data of the substrate obtained by the above calculations were reduced in dimension and visualized for bacteria classification. Figure 8 This is a bacterial species classification diagram of the bacteria in Example 4 of the present disclosure.

[0108] like Figure 8 As shown, by using dimensionality reduction analysis technology, high-dimensional data is converted into low-dimensional data, while retaining important difference information, achieving bacterial typing and distinguishing the three types of bacteria. It shows that the characteristic bar graph based on the conversion degree data provided by the present disclosure can distinguish and identify the three types of bacteria.

[0109] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above description is only a specific embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A method for identifying bacterial species by portable mass spectrometry based on substrate-enzyme reaction, comprising: Cultivating the bacteria extracted from the sample to be tested to obtain a bacterial culture solution of the sample to be tested, and washing and diluting the bacterial culture solution of the sample to be tested to obtain a bacterial suspension of the sample to be tested; Adding a plurality of preselected substrates to the bacterial suspension of the sample to be tested for co-incubation, so that the bacterial suspension of the sample to be tested and the preselected substrates undergo an enzyme reaction to obtain a bacterial substrate mixed solution containing an enzyme reaction product; Using electrospray mass spectrometry to detect the bacterial substrate mixture, obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product, and calculate the conversion degree data for each preselected substrate; According to the conversion degree data of the plurality of preselected substrates, drawing a characteristic column map of bacteria in the sample to be tested in a coordinate system based on the preselected substrate types and conversion degrees; The characteristic column map of bacteria in the sample to be tested is matched with the characteristic column map in the bacterial species database to identify the species of bacteria in the sample to be tested.

2. The method according to claim 1, characterized in that: The method for establishing the bacterial species database comprises: For different pure strains of known bacteria, the operations of culturing, co-incubation, mass spectrometry detection and mapping are performed to obtain characteristic column maps of different pure strains of known bacteria to form a bacterial species database. The known pure bacteria include any one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacteriaceae.

3. The method according to claim 2, further comprising: When the bacterial species extracted from the sample to be tested is not included in the bacterial species database, after the bacterial species is determined in combination with other bacterial species identification methods, the characteristic column map of the bacteria extracted from the sample to be tested is added to the bacterial species database.

4. The method according to claim 1, characterized in that The preselected substrates include any two or more of L-glutamyl-3-carboxyl-4-nitroaniline, L-proline aniline, 4-aminophenyl-β-D-galactoside, L-tyrosine-4-nitroaniline, L-pyroglutamic acid-2-naphthylamide, phenyl-2-acetylamino-2-deoxy-α-D-pyranoglucopyranoside, and L-alanine-4-nitroanilide, preferably 2 to 5; The preselected substrate concentration is 0.1-10 ppm, and the dosage is 0.5-40 μL.

5. The method according to claim 1, characterized in that The culture medium includes any one of agar medium, nutrient broth medium, and LB liquid medium; The culture time is 8 to 16 hours, and the culture temperature is 25°C to 40°C; The concentration of the bacterial suspension was 10 7 ~10 9 cells / mL.

6. The method according to claim 1, characterized in that The co-incubation temperature is 25°C to 45°C; The co-incubation time is 5 min to 240 min, preferably 5 min to 30 min.

7. The method according to claim 1, characterized in that The method of performing mass spectrometry detection on the bacterial substrate mixed solution by electrospray mass spectrometry comprises: The bacterial substrate mixed solution is injected into a capillary and an electrospray solvent is added, a voltage is applied to the capillary to generate an ionized spray, and the ionized spray is detected by mass spectrometry to obtain the mass spectrometry signal intensity of the preselected substrate and the enzyme reaction product.

8. The method according to claim 7, characterized in that The capillary diameter is 1-10 μm; The applied voltage is a direct current voltage or an alternating current voltage, and the voltage magnitude is 1.5-5 kV; The spray solvent is an organic solvent, including at least one of methanol, acetonitrile and acetone.

9. The method according to claim 1, further comprising: The conversion degree data of the substrate are subjected to dimension reduction and visualization analysis to classify different types of bacteria.

10. The method according to claim 1, characterized in that The sample to be tested includes at least one of whole blood, cell-infected tissue, and urine.

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