Method and device for detecting burkholderia gladioli
Through mass spectrometry detection technology, MALDI-TOF MS was used to detect protein samples of Cyclotridium gladiolus, and the properties of the strain were judged based on the characteristic peak mass-to-mass-to-charge ratio in the mass spectrometry peak map, solving the cumbersome and time-consuming problem in the existing technology, and achieving a fast and efficient detection effect.
Patent Information
- Application Number
- CN202311823093.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-27
- Publication Date
- 2025-06-27
AI Technical Summary
The methods for detecting Cyclotridium gladiolus in the prior art are cumbersome and time-consuming, and rapid detection in food safety cannot be achieved.
Mass spectrometry detection technology was used to detect protein samples of the strain to be tested by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), mass spectrometry peak map was obtained, and whether the strain was a toxin-producing strain was determined based on the mass-to-charge ratio of the characteristic peaks.
Fast, efficient and low-cost detection of whether Cyclotridium gladiolus is a toxin-producing strain is achieved, significantly shortening the detection time.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of mass spectrometry analysis, and particularly to a method and a device for detecting Burkholderia gladioli. Background Art
[0002] Burkholderia gladioli, a Gram-negative bacterium, is facultatively anaerobic and widely exists in nature. During the natural fermentation of substances such as fermented rice noodles, it is extremely vulnerable to contamination by Burkholderia gladioli, mainly because Burkholderia gladioli can produce bongkrekic acid, toxin B, etc. After humans accidentally ingest food contaminated with Burkholderia gladioli, they may show severe clinical symptoms, and the clinical mortality rate exceeds 40%.
[0003] Currently, the main method for identifying whether a strain is Burkholderia gladioli is the traditional culture method (refer to the national standard GB / T 4789.29-2020): After the sample is cultured in GVC enrichment broth at 36±1°C for 20h to 24h, it is separated onto different isolation plates, and after culturing for 24h to 48h, the characteristics such as the size, shape, and color of the colonies are observed. Subsequently, typical or suspected colonies are inoculated onto egg yolk agar plates, and after culturing for 24h, preliminary screening tests such as Gram staining and oxidase tests are carried out, and then transferred to PDA plates for culturing for 24h and biochemical tests, serotype typing, and toxicity tests are carried out. The toxicity test takes 7 days to obtain negative or positive results for virulence determination. Just from culturing to identifying whether a strain is Burkholderia gladioli, the traditional culture method requires 3 to 4 days. If it is necessary to further determine whether it is a toxin-producing strain, it takes 5 to 7 days. The entire detection process has cumbersome steps and a long detection time, and cannot achieve rapid detection in food safety.
[0004] Therefore, the method for detecting Burkholderia gladioli urgently needs to be improved. Summary of the Invention
[0005] Based on this, one or more embodiments of the present application provide a method and a device for rapidly and efficiently detecting Burkholderia gladioli. The technical solutions include:
[0006] According to the first aspect of the embodiments of the present application, a method for detecting Burkholderia gladioli is provided, including the following steps:
[0007] Provide a protein sample of the strain to be tested, perform mass spectrometry detection, and obtain the mass spectrometry peak map of the strain to be tested;
[0008] If the mass spectrometry peak map contains a first characteristic peak, the strain to be tested includes strains other than toxin-producing strains;
[0009] If the mass spectrometry peak map contains a second characteristic peak, the strain to be tested includes a toxin-producing strain;
[0010] The mass-to-charge ratio of the first characteristic peak is 7207 Da to 7211 Da, and the mass-to-charge ratio of the second characteristic peak is 7716 Da to 7721 Da.
[0011] In one embodiment, the toxin includes one or more of bongkrekic acid and toxoflavin.
[0012] In one embodiment, the method for mass spectrometry detection includes matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.
[0013] In one embodiment, the parameters of the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry include: the detection mass range is 2000 Da to 20000 Da, the acceleration voltage is 20000 V to 22500 V, the focusing voltage is 5000 V to 5500 V, the detection voltage is 1600 V to 1800 V, the number of laser pulses is 208 to 400 times, and the laser energy is 5 μJ to 8 μJ.
[0014] In one embodiment, it includes the following steps:
[0015] Inoculate the strain to be tested into a culture medium to obtain single colonies; and
[0016] Apply the single colonies to the surface of the target plate;
[0017] Sequentially add a lysis solution and a sample matrix solution to the target plate coated with single colonies, air-dry, and prepare a protein sample of the strain to be tested.
[0018] In one embodiment, the lysis solution includes formic acid.
[0019] In one embodiment, the sample matrix solution includes α-cyano-4-hydroxycinnamic acid.
[0020] According to the second aspect of the embodiments of the present application, a device for detecting Burkholderia gladioli is provided, including:
[0021] A data acquisition module for acquiring the mass spectrometry peak map of the strain to be tested; and
[0022] A data analysis module for analyzing whether the strain to be tested includes a toxin-producing strain according to the mass spectrometry peak map; if the mass spectrometry peak map contains a first characteristic peak, the strain to be tested includes a strain other than the toxin-producing strain; if the mass spectrometry peak map contains a second characteristic peak, the strain to be tested includes a toxin-producing strain.
[0023] According to a third aspect of the embodiments of the present application, there is provided a computer device, including a memory and a processor, where the memory stores a computer program, and when the processor executes the computer program, the steps of the above method are implemented.
[0024] According to a fourth aspect of the embodiments of the present application, there is provided a computer-readable storage medium storing a computer program, and when the computer program is executed by a processor, the steps of the above method are implemented.
[0025] Compared with the traditional technology, the present application has the following beneficial effects:
[0026] The applicant of the present application studied a large number of toxin-producing strains and non-toxin-producing strains and found that all Burkholderia gladioli strains carrying characteristic peaks with a mass-to-charge ratio of 7716 Da to 7721 Da can produce toxins, while strains carrying characteristic peaks with a mass-to-charge ratio of 7207 Da to 7211 Da do not produce toxins. Based on this, the present application provides a method capable of quickly, efficiently, and low-costly detecting whether Burkholderia gladioli is a toxin-producing strain. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0028] Figure 1 It is a schematic flowchart for detecting Burkholderia gladioli in an embodiment of the present application;
[0029] Figure 2 It is a mass spectrometry characteristic peak diagram of 5 toxin-producing strains in Embodiment 1 of the present application;
[0030] Figure 3 It is a mass spectrometry characteristic peak diagram of 6 non-toxin-producing strains in Embodiment 1 of the present application;
[0031] Figure 4 It is a comparison diagram of the first characteristic peaks of toxin-producing strains and non-toxin-producing strains;
[0032] Figure 5 It is a comparison diagram of the second characteristic peaks of toxin-producing strains and non-toxin-producing strains;
[0033] Figure 6 It is a mass spectrometry characteristic peak diagram of Sample 1 and Sample 2 in Embodiment 2 of the present application;
[0034] Figure 7 It is a mass spectrometry characteristic peak diagram of Sample 3 and Sample 5 in Embodiment 2 of the present application. Detailed implementation manners
[0035] To make the above objects, features, and advantages of the present application more obvious and understandable, the detailed implementation manners of the present application will be described in detail. Many specific details are set forth in the following description to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.
[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in this application can be obtained through the market or prepared by existing methods.
[0037] The selection scope of the terms "and / or", "or / and", and "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", and "and / or" are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").
[0038] In this application, the terms "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specifically defined, refer to a quantity greater than 2 or equal to 2. For example, "one or more types" means one type or greater than or equal to two types.
[0039] The "combinations thereof", "any combinations thereof", "any combination manners thereof", etc. used herein include all suitable combination manners of any two or any two or more of the listed items.
[0040] In this application, the "suitable combination method", "suitable method", "any suitable method", etc., the "suitable" described therein shall be based on being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.
[0041] In this application, "preferred", "better", "more preferable", "preferably" are only used to describe the implementation manners or embodiments with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.
[0042] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.
[0043] In this application, "optionally", "optional", "option", mean that it can be either present or absent, that is, it refers to any one of the two parallel options of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.
[0044] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.
[0045] In this application, regarding the numerical interval (that is, the numerical range), without special instructions, the optional values are considered continuous within the above numerical interval, and include the two numerical endpoints (that is, the minimum value and the maximum value) of this numerical range, as well as each numerical value between these two numerical endpoints. Without special instructions, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 - 10, which means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions for features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.
[0046] Some embodiments of this application provide a method for detecting Burkholderia gladioli, including steps S10 to S20.
[0047] Step S10: Provide a protein sample of the strain to be tested, perform mass spectrometry detection, and obtain the mass spectrometry peak map of the strain to be tested.
[0048] In some of these embodiments, in step S10, the following steps are included: pretreating the strain to be tested to obtain a protein sample, performing mass spectrometry detection, and obtaining a mass spectrometry peak map of the strain to be tested.
[0049] In some of these embodiments, in step S10, the method for performing mass spectrometry detection includes matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).
[0050] In some of these embodiments, in step S10, the parameters of MALDI-TOF MS include: the detection mass range is 2000 Da to 20000 Da, the acceleration voltage is 20000 V to 22500 V, the focusing voltage is 5000 V to 5500 V, the detection voltage is 1600 V to 1800 V, the number of laser pulses is 208 to 400 times, and the laser energy is 5 μJ to 8 μJ.
[0051] Optionally, the parameters of MALDI-TOF MS include: the detection mass range is 2000 Da to 20000 Da, the acceleration voltage is 21000 V, the focusing voltage is 5000 V, the detection voltage is 1650 V, the number of laser pulses is 256 times, and the laser energy is 6 μJ.
[0052] In some other embodiments, the instrument parameters can also be other values that conform to the YY1740.2-2021 standard.
[0053] In some of these embodiments, in step S10, the steps of the pretreatment include steps S11 to S13.
[0054] Step S11: Inoculate the strain to be tested into a culture medium to obtain a single colony.
[0055] In some specific embodiments, the above single colony can be an isolated and purified single colony or an unisolated and unpurified single colony.
[0056] In some specific embodiments, in step S11, the above culture medium includes one of potato dextrose agar (PDA), blood agar medium, and modified potato dextrose agar (mPDA).
[0057] In some specific embodiments, in step S11, the culture temperature is 27°C to 29°C.
[0058] In some specific embodiments, in step S11, the culture time is 20 h to 24 h.
[0059] Step S12: Spread the single colony onto the surface of the target plate.
[0060] Step S13: Add lysis buffer to the target plate smeared with single colonies. After air-drying, add sample matrix solution, and then air-dry to prepare the sample to be tested.
[0061] In some embodiments, the lysis buffer includes formic acid and water.
[0062] In some embodiments, the sample matrix solution is selected from α-cyano-4-hydroxycinnamic acid matrix (CHCA).
[0063] In some specific embodiments, the addition amount of the lysis buffer to the target plate is 0.8 μL to 1.2 μL. Optionally, the addition amount of the lysis buffer is 1.0 μL.
[0064] In some specific embodiments, the addition amount of the sample matrix solution to the target plate is 0.8 μL to 1.2 μL. Optionally, the addition amount of the sample matrix solution is 1.0 μL.
[0065] Understandably, after fixing single cells on the target plate of the mass spectrometer, adding the lysis buffer and the sample matrix solution can fully precipitate proteins to obtain more protein samples.
[0066] In some embodiments, in step S10, the pre-treatment steps include the following steps:
[0067] Inoculate the strain to be tested into a culture medium to obtain single colonies;
[0068] Smear the single colonies onto the target points on the surface of the target plate;
[0069] Add sample matrix solution to the target plate smeared with single colonies, and air-dry to prepare the sample to be tested.
[0070] In some embodiments, in step S10, the pre-treatment steps include the following steps:
[0071] Disperse the colonies in 75% ethanol for inactivation, let stand for 5 min, centrifuge to discard the supernatant, add lysis buffer I (the main component is formic acid) to the precipitate, shake and mix well, then let stand for 5 min, add an equal volume of lysis buffer II (the main component is acetonitrile), shake and mix well, then centrifuge, take 1 μL of the supernatant and add it to the target point, dry, and finally drop the sample matrix solution and dry.
[0072] In some embodiments, in step S10, steps S14 to S15 are further included.
[0073] Step S14: Calibrate the mass axis of the mass spectrometry detector using a calibrator.
[0074] Step S15: Send the target plate coated with the strain sample to be tested into the injection chamber for detection to obtain the mass spectrometry peak pattern of the strain to be tested.
[0075] Traditional methods for detecting Burkholderia gladioli involve strain cultivation, isolation and purification, morphological observation, biochemical identification, and toxin production tests. From strain cultivation to reporting, it takes about 10 to 11 days. The test process is cumbersome and time-consuming, and rapid detection cannot be achieved. However, based on MALDI-TOF MS technology, this application can detect single colonies without isolation and purification, and it only takes 1 to 2 days from strain cultivation to reporting, significantly accelerating the detection process.
[0076] Step S20: According to the mass spectrometry peak pattern of the strain to be tested, analyze whether the strain to be tested includes toxin-producing strains.
[0077] In some embodiments, in step S20, if the mass spectrometry peak pattern of the strain to be tested contains a first characteristic peak, then the strain to be tested includes strains other than toxin-producing strains.
[0078] Specifically, the mass-to-charge ratio of the first characteristic peak is 7207 Da to 7211 Da. Optionally, the mass-to-charge ratio of the first characteristic peak is 7209 Da.
[0079] In some embodiments, in step S20, if the mass spectrometry peak pattern of the strain to be tested contains a second characteristic peak, then the strain to be tested includes toxin-producing strains.
[0080] Specifically, the mass-to-charge ratio of the second characteristic peak is 7716 Da to 7721 Da. Optionally, the mass-to-charge ratio of the second characteristic peak is 7719 Da.
[0081] It is understandable that the strains other than toxin-producing strains in this application refer to Burkholderia gladioli that cannot produce toxins.
[0082] In some embodiments, toxin-producing strains include strains that produce one or more toxins such as bongkrekic acid and toxoflavin. In other embodiments, toxin-producing strains also include strains that produce other types of toxins.
[0083] Under suitable temperature and humidity conditions, Burkholderia gladioli can produce large amounts of toxins such as bongkrekic acid and toxoflavin in a short time. If humans ingest it, it will cause severe food poisoning and even death. Under the same conditions, the production amount of bongkrekic acid is much larger than that of toxoflavin, and its toxicity is stronger. Bongkrekic acid is the main cause of poisoning.
[0084] In some embodiments, if the mass spectrometry peak pattern of the strain to be tested includes the above-mentioned first characteristic peak, then the strain to be tested cannot produce toxins, that is, the strain to be tested is a strain other than toxin-producing strains (non-toxin-producing strain).
[0085] In some of these embodiments, if the mass spectrometry peak pattern of the strain to be tested includes the above-mentioned second characteristic peak, then the strain to be tested can produce toxins, that is, the strain to be tested is a toxin-producing strain (toxin-producing strain).
[0086] In some of these embodiments, if the mass spectrometry peak pattern of the strain to be tested neither includes the above-mentioned first characteristic peak or the above-mentioned second characteristic peak, or if the mass spectrometry peak pattern of the strain to be tested includes both the above-mentioned first characteristic peak and the above-mentioned second characteristic peak at the same time, then other methods need to be used for further confirmation.
[0087] In some of these embodiments, the method for detecting Burkholderia gladioli further includes the following steps: analyzing whether the strain to be tested is Burkholderia gladioli through an identification system.
[0088] Optionally, the identification system includes at least one of the Hexin Kangyuan full-automatic microbial mass spectrometry detection system (CMI-1600), the bioMérieux full-automatic rapid microbial mass spectrometry detection system VITEK MS, and the Bruker full-automatic rapid bio-mass spectrometry detection system (Bruker MALDI Biotyper).
[0089] In some of these embodiments, the flow of the method for detecting Burkholderia gladioli is as Figure 1 shown.
[0090] The above detection method of the present application has the advantages of being fast, efficient, low-cost, and high-throughput; according to the detection results of the present application, food safety can be rapidly screened. On the one hand, the method of the present application can be used for rapid identification of Burkholderia gladioli in food; on the other hand, the method of the present application can classify toxin-producing strains and non-toxin-producing strains in Burkholderia gladioli.
[0091] A device for detecting Burkholderia gladioli includes:
[0092] A data acquisition module for acquiring the mass spectrometry peak pattern of the strain to be tested; and
[0093] A data analysis module for analyzing whether the strain to be tested includes a toxin-producing strain according to the mass spectrometry peak pattern; if the mass spectrometry peak pattern contains a first characteristic peak, then the strain to be tested includes strains other than the toxin-producing strain; if the mass spectrometry peak pattern contains a second characteristic peak, then the strain to be tested includes a toxin-producing strain.
[0094] It is understandable that each module in the above device can be implemented in whole or in part by software, hardware, and their combination. The above modules can be embedded in or independent of the processor in the computer device in the form of hardware, or can be stored in the memory of the computer device in the form of software, so that the processor can call and execute the operations corresponding to the above modules.
[0095] A computer device includes a memory and a processor. The memory stores a computer program, and when the processor executes the computer program, the steps of the above detection method are implemented.
[0096] A computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the steps of the above detection method are implemented.
[0097] The present application will be further described below in conjunction with specific embodiments and comparative examples, but it should not be construed as a limitation on the protection scope of the present application. For the raw materials involved in the following specific embodiments, unless otherwise specified, they can all be obtained commercially. For the instruments used, unless otherwise specified, they can all be obtained commercially. For the processes involved, unless otherwise specified, they are all conventional selections of those skilled in the art.
[0098] Example 1:
[0099] (1) Provide 11 Burkholderia gladioli isolates (from Guangzhou Inspection, Testing and Certification Group), and identify the 11 isolates with reference to the GB4789.29-2020 standard and the GB5009.189-2016 standard, including 6 non-toxigenic strains (A1 to A6) and 5 toxigenic strains (B1 to B5).
[0100] (2) Strain culture: Inoculate the above 11 isolates into PDA medium and culture at 28 °C for 24 h to grow single colonies on the medium.
[0101] (3) Pretreatment: Pick a single colony with an inoculation loop, evenly smear it on the target plate of MALDI-TOF MS, and add 1 μL of lysis solution (mainly composed of formic acid and water); after drying, add 1 μL of CHCA (α-cyano-4-hydroxycinnamic acid) matrix; after drying, send the target plate into the sample injection chamber for detection. Set 3 biological replicates for each strain.
[0102] (4) Mass spectrometry detection: Detect the strains to be tested by MALDI-TOF MS, and repeat the detection of each strain sample 3 times. After calibrating the mass axis with a calibrator, detect the samples, and the main parameters are shown in Table 1.
[0103] Table 1
[0104] Item Parameter Mass range 2000Da - 20000Da Acceleration voltage 22100V Focusing voltage 5000V Detection voltage 1650V Number of laser strikes 256 times Laser energy 6μJ
[0105] Group the mass spectrometry peak maps of the toxigenic strains and non-toxigenic strains, namely the toxigenic group and the non-toxigenic group. Set the signal-to-noise ratio parameter of the mass spectrometry peak maps of all strains to be tested to 3, extract the characteristic peaks of all mass spectrometry peak maps in the toxigenic group, and obtain the toxigenic strain spectrum as shown in Figure 2 shown and as shown in Figure 3The spectrum diagram of the non-toxin-producing strain shown.
[0106] Furthermore, by comparing and analyzing the characteristic peaks of the toxin-producing strain and the non-toxin-producing strain, it was found that all Burkholderia gladioli strains carrying characteristic peaks with a mass-to-charge ratio of 7207 Da to 7211 Da (the first characteristic peak) were non-toxin-producing strains, while all strains carrying characteristic peaks with a mass-to-charge ratio of 7716 Da to 7721 Da (the second characteristic peak) were toxin-producing strains. The statistical results of the non-toxin-producing strains are shown in Table 2, and the statistical results of the toxin-producing strains are shown in Table 3. Other characteristic peaks may exist in both strains and cannot be used to distinguish between toxin-producing and non-toxin-producing strains.
[0107] Table 2
[0108]
[0109]
[0110] Table 3
[0111]
[0112]
[0113] The comparison of the characteristic peaks of the toxin-producing and non-toxin-producing strains at a mass-to-charge ratio of about 7209 Da is as Figure 4 shown; the comparison of the characteristic peaks at a mass-to-charge ratio of about 7719 Da is as Figure 5 shown. The above results indicate that in this application, by detecting the protein fingerprint of an unknown strain based on the MALDI-TOF MS technology, it is possible to quickly identify whether the strain is Burkholderia gladioli; and based on the characteristic peaks of the strain, it is possible to quickly distinguish between the toxin-producing and non-toxin-producing strains of Burkholderia gladioli.
[0114] Example 2:
[0115] The detection method of this application was used to verify 3 known toxin-producing strains and 3 known non-toxin-producing strains of Burkholderia gladioli (Samples 1 to 6). The specific steps are as follows:
[0116] (1) The above 6 isolates were respectively inoculated into PDA medium and cultured at 28 °C for 24 h.
[0117] (2) Use an inoculation loop to pick a single colony and evenly smear it on the target plate of MALDI-TOF MS, and add 1 μL of lysis solution (the main components are formic acid and water); after drying, add 1 μL of CHCA matrix; after drying, send the target plate into the injection chamber for detection.
[0118] (3) Detect the strain to be tested by MALDI-TOF MS. The mass spectrometry information of the above strains is shown in Table 4. The mass spectrometry peak maps of Sample 1 and Sample 2 are as shown in Figure 6 shown, and the mass spectrometry peak maps of Sample 3 and Sample 5 are as shown in Figure 7 shown. The first characteristic peak and the second characteristic peak are not detected in Sample 4 and Sample 6.
[0119] Table 4
[0120]
[0121] The above results show that the method of this application for classifying toxin-producing strains and non-toxin-producing strains of Burkholderia gladioli has the advantages of high speed and accuracy.
[0122] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.
[0123] The above embodiments only represent several implementation manners of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. Therefore, the protection scope of this application should be subject to the appended claims.
Claims
1. A method for detecting Burkholderia gladioli, characterized in that, It includes the following steps: Provide a protein sample of the strain to be tested, perform mass spectrometry detection, and obtain the mass spectrometry peak map of the strain to be tested; If the mass spectrometry peak map contains a first characteristic peak, the strain to be tested includes strains other than toxin-producing strains; If the mass spectrometry peak map contains a second characteristic peak, the strain to be tested includes toxin-producing strains; The mass-to-charge ratio of the first characteristic peak is 7207 Da to 7211 Da, and the mass-to-charge ratio of the second characteristic peak is 7716 Da to 7721 Da.
2. The method for detecting Burkholderia gladioli according to claim 1, wherein The toxin includes one or more of bongkrekic acid and toxoflavin.
3. The method for detecting Burkholderia gladioli according to any one of claims 1 to 2, characterized in that, The method of the mass spectrometry detection includes matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
4. The method for detecting Burkholderia gladioli according to claim 3, wherein The parameters of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry include: the detection mass range is 2000 Da to 20000 Da, the acceleration voltage is 20000 V to 22500 V, the focusing voltage is 5000 V to 5500 V, the detection voltage is 1600 V to 1800 V, the number of laser pulses is 208 to 400 times, and the laser energy is 5 μJ to 8 μJ.
5. The method for detecting Burkholderia gladioli according to any one of claims 1 to 2 and 4, characterized in that, It includes the following steps: Inoculate the strain to be tested into a culture medium to obtain single colonies; And Apply the single colonies to the surface of the target plate; Sequentially add a lysis solution and a sample matrix solution to the target plate coated with single colonies, air-dry, and prepare a protein sample of the strain to be tested.
6. The method for detecting Burkholderia gladioli according to claim 5, wherein The lysis solution includes formic acid.
7. The method for detecting Burkholderia gladioli according to claim 5, wherein The sample matrix solution includes α-cyano-4-hydroxycinnamic acid.
8. A device for detecting Burkholderia gladioli, characterized in that, It includes: A data acquisition module for acquiring the mass spectrometry peak map of the strain to be tested; And A data analysis module for analyzing whether the strain to be tested includes toxin-producing strains according to the mass spectrometry peak map; if the mass spectrometry peak map contains a first characteristic peak, the strain to be tested includes strains other than toxin-producing strains; if the mass spectrometry peak map contains a second characteristic peak, the strain to be tested includes toxin-producing strains.
9. A computer device, comprising a memory and a processor, the memory storing a computer program, characterized in that, When the processor executes the computer program, it implements the steps of the method according to any one of claims 1 to 7.
10. A computer-readable storage medium storing a computer program, characterized in that, When the computer program is executed by the processor, it implements the steps of the method according to any one of claims 1 to 7.