CN113380331A - Burkholderia melioidis identification spectrum database and quick identification method of Burkholderia melioidis - Google Patents
Burkholderia melioidis identification spectrum database and quick identification method of Burkholderia melioidis Download PDFInfo
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Abstract
The invention provides a burkholderia farinae Boeck Hall bacterium identification spectrum database and a quick identification method of the burkholderia farinae Boeck Hall bacterium, wherein the database comprises a protein map with the molecular weight of 2000-20000 of the burkholderia farinae Boeck bacterium, and the protein map is formed by drawing the mass-to-mass ratio and the relative strength of proteins with different molecular weights; the invention also discloses data of 38 characteristic peaks in the map. The invention discloses a method for rapidly identifying Burkholderia melioidis, which comprises the following steps: 1) inoculating the detected bacteria to a flat plate, and culturing the inoculated flat plate in a constant temperature box at 35-37 ℃ for 20-48 h; 2) selecting a single colony on a plate, coating a target plate, adding a matrix solution immediately after a mycoderm is dried in the air, and detecting by using MALDI-TOF MS after the mycoderm is dried in the air; comparing the obtained map with the maps in the database, and judging the map to be positive when Log (score) is more than or equal to 2.0. By adopting the map database and the rapid identification method, the identification accuracy of the burkholderia melioidis reaches more than 99.9 percent, and the rapid and accurate identification of the burkholderia melioidis is realized.
Description
Technical Field
The invention relates to the technical field of bacteria identification, in particular to a burkholderia farinae identification spectrum database and a quick identification method of burkholderia farinae.
Background
The natural epidemic origin of Burkholderia Pseudonarum (BP) is mainly in tropical and subtropical regions, the farcinia pseudonarum epidemic area in China is mainly in the southern region, the most common farcinia pseudonarum in the tropical Henan is the tropical, the natural epidemic area has the characteristics of multiple drug resistance, easy dispersion, atypical infection symptoms and the like, the natural epidemic area becomes a frequently encountered disease and a common disease in the Henan tropical region, the mortality rate of the disease is up to 90% reported by related documents, and the mortality rate of the disease is still up to 60% along with the development of antibiotics and the progress of inspection technology; among them, BP has the highest separation rate in tropical Hainan, and BP can be a new type of tropical diseases. The rapid diagnosis of BP infection is the key to the treatment and prevention of melioidosis, and the inaccurate result of conventional biochemical identification can cause the wrong report and the missed report.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology is a novel soft ionization biological mass spectrum developed in recent years. Compared with other methods, the pathogenic bacteria identification by using the MALDI-TOF MS technology is faster, more convenient and more accurate, and the cost is saved. The MALDI-TOF-MS technology has good application effect in the rapid identification of Listeria monocytogenes (Listeria monocytogenes), Streptococcus pneumoniae (Streptococcus pneumoniae), Bacteroides fragilis (Bacteroides fragilis), Neisseria meningitidis (Neisseria meningitidis), Salmonella (Salmonella sp.), Enterobacter sakazakii (Enterobacter sakazakii) and plant pathogenic bacteria. For example, patent application publication No. CN104833803B discloses a multiplex detection method and database for pathogenic bacteria of legumes, which discloses map information of the database and uses the constructed database to detect 7 important pathogenic bacteria of legumes. The method realizes identification of plant pathogenic bacteria. Bacterial proteins are largely dependent on the genetic factors of the bacteria themselves and therefore vary in the detection results for different species. There is also a document "identification of Burkholderia meliloti and Burkholderia pseudomeliloti by 3 methods" which confirms that MALDI-TOF-MS can be used for identification of BP. The document compares the strain to be detected with a self-established pathogen protein fingerprint database, does not disclose the self-established database data, and needs to improve the detection accuracy.
The MALDI-TOF-MS technology takes bacterial mycoprotein as a detection object, and the accuracy of an identification result is influenced by a protein fingerprint in a database. Currently, there are few relevant studies for the identification of BP for MALDI-TOF-MS and there is a lack of corresponding standard protein profiling databases. This greatly restricts the application of MALDI-TOF-MS technology to clinical BP identification.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a burkholderia farci identification spectrum database and a burkholderia farci rapid identification method.
The technical scheme of the invention is realized as follows:
an identification spectrum database of burkholderia farci comprises a protein spectrum of the burkholderia farci with molecular weight of 2000-20000, wherein the protein spectrum is formed by drawing the mass-to-mass ratio and the relative strength of proteins with different molecular weights;
the protein with the molecular weight of 2000-3000 has 3 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 2190.8179, 2697.1714, 2840.8544;
the protein with the molecular weight of 3000-4000 has 9 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 3132.8089, 3210.5085, 3144.2096, 3210.5085, 3278.7055, 3583.1600, 3640.8447, 3856.7721, 3938.5076;
the protein with the molecular weight of 4000-6000 has 7 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 4366.7200, 4451.0223, 4769.6272, 4778.9532, 5070.4933, 5348.3594, 5381.5558;
the protein with the molecular weight of 6000-8000 has 7 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 6255.3870, 6316.1295, 6387.0116, 7158.8015, 7274.6620, 7708.2632, 7872.5290;
the protein with the molecular weight of 8000-10000 has 6 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 8328.4155, 8880.0214, 9067.7875, 9230.8977, 9560.6676, 9746.2004;
the protein with the molecular weight of 10000-12000 has 4 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 10145.7753, 10308.1668, 10704.8187, 11234.7706;
the protein with the molecular weight of 12000-14000 has 2 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 12224.8512, 13026.6093.
The invention also provides a method for rapidly identifying Burkholderia meliloti, which comprises the following steps:
1) inoculating the detected bacteria to a flat plate, and culturing the inoculated flat plate in a constant temperature box at 35-37 ℃ for 20-48 h;
2) selecting a single colony on a plate, coating a target plate, adding a matrix solution immediately after a mycoderm is dried in the air, and detecting by using MALDI-TOF MS after the mycoderm is dried in the air; comparing the obtained detected bacteria map with the map in the database of claim 1, and judging the bacteria map to be positive when the log (score) is more than or equal to 2.0.
Preferably, the matrix solution is a 12-15 mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving a basic solvent; the basic solvent contains acetonitrile with volume final concentration of 45-55%, trifluoroacetic acid with volume final concentration of 2.3-2.8% and the balance of water.
Preferably, the matrix solution is a 14mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving a basic solvent; the base solvent contained acetonitrile at a final volume concentration of 50%, trifluoroacetic acid at a final volume concentration of 2.5%, and the balance water.
Preferably, the plate is a columbian blood plate or a chocolate plate.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes a BP identification map database which can be applied to rapid (5-10 minutes) identification of clinical BP, wins time for selecting effective drugs to treat patients and can reduce mortality.
The invention also provides a method for rapidly identifying Burkholderia melioidis, provides a standard operation process, determines the optimal bacterial culture medium and culture conditions, and avoids the influence of the factors on the identification result. By adopting the map database and the rapid identification method, the identification accuracy of BP reaches more than 99.9 percent, and the rapid and accurate identification of BP is realized.
By adopting the database and the identification method, the burkholderia melioidea can be distinguished from the relative burkholderia cepacia and burkholderia melioidea.
Drawings
FIG. 1: molecular weight at 2000-3000
FIG. 2: molecular weight at 3000-4000
FIG. 3: molecular weight 4000-6000 spectrum
FIG. 4: molecular weight 6000-8000 spectrum
FIG. 5: molecular weight 8000-10000 spectrum
FIG. 6: map of molecular weight 10000-
FIG. 7: map of molecular weight 12000-14000
FIG. 8: identification map (general chart) of Bokholderia farci
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1: creation of a database
1. The experimental scheme is as follows:
1.1, experimental reagent: 70% formic acid solution, pure acetonitrile, α -cyano-4-hydroxyphenylacrylic acid (HCCA), trifluoroacetic acid (TFA), all from Merrier, France.
1.2, main experimental instruments: matrix assisted laser desorption ionization time-of-flight mass spectrometer (VITEK MS) (merriey, a biological france).
1.3, experimental method:
pretreatment of MALDI-TOF MS strain: collecting multiple strains of clinical infected Boeck farinosus, inoculating Boeck farinosus strain to Columbia blood agar plate, placing at 35 deg.C and 5% CO2Culturing in incubator for 24 hr, selecting appropriate amount of bacterial colony with sterilized bacteria-taking ring, and coating on targetOn the plate, the calibration wells are coated with ATCC8739 strain as quality control, 1 mul of matrix solution is dripped to uniformly cover the plates, and the plates are naturally dried for later use. The target plate is placed into a MALDI-TOF mass spectrometer for on-machine detection (N2 laser: 337 nm; ion source: 20.0 KV; detection mode: linear positive ion; laser frequency: 50 HZ; laser energy: 64), the identification software VITEK MS of MALDI-TOF MS is adopted for sample data acquisition and identification, and a protein map with molecular weight of 2000-20000Da is acquired. The protein mass spectrum of each sample is obtained by 100 times of laser clicks at different positions, and the collected protein peak information is corrected by software. Protein peak information was obtained by VITEK MS RUO software, analyzed and an identification database was established.
The comparison analysis and the experiment prove that the protein identification characteristic peak data of the burkholderia melioidis are determined:
the protein with the molecular weight of 2000-3000 has 3 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 2190.8179, 2697.1714, 2840.8544;
the protein with the molecular weight of 3000-4000 has 9 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 3132.8089, 3210.5085, 3144.2096, 3210.5085, 3278.7055, 3583.1600, 3640.8447, 3856.7721, 3938.5076;
the protein with the molecular weight of 4000-6000 has 7 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 4366.7200, 4451.0223, 4769.6272, 4778.9532, 5070.4933, 5348.3594, 5381.5558;
the protein with the molecular weight of 6000-8000 has 7 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 6255.3870, 6316.1295, 6387.0116, 7158.8015, 7274.6620, 7708.2632, 7872.5290;
the protein with the molecular weight of 8000-10000 has 6 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 8328.4155, 8880.0214, 9067.7875, 9230.8977, 9560.6676, 9746.2004;
the protein with the molecular weight of 10000-: 10145.7753, 10308.1668, 10704.8187, 11234.7706;
the protein with the molecular weight of 12000-14000 has 2 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 12224.8512, 13026.6093.
The identification map (the characteristic peak data of the molecular weight detection range 2000-. And comparing the atlas of the strain to be detected with the database constructed by the invention, and determining that the strain to be detected is Boeck farinosa Hold when Log (score) is more than or equal to 2.0. The accuracy rate of the research database identification reaches 99.90%. Table 1 below provides the identification results of several strains.
TABLE 1
| Bacterial species name | Log(score) |
| Bokeh fungus (Standard strain) farcina | 2.5 |
| Burkholderia cepacia (Standard strain) | 1.8 |
| Bokehol fungus (Standard strain) for gangrene | 1.7 |
The results show that the Burkholderia melioidea can be distinguished from the relative Burkholderia cepacia and Burkholderia melioidea (Burkholderia malili) by applying the database provided by the invention. The standard bacterial strain of Boeck hollandis farreri identified by the identification of the bacterial strain database is collected and identified by the database of the invention, and the accuracy rate reaches more than 99.90 percent.
The identification operation method also influences the acquisition of map data and the accuracy of the identification result. The following examples provide comparative results of identification using different media and incubation times.
Example 2: optimization of culture conditions of the strains
The method comprises the following steps: the method for rapidly identifying Burkholderia melioidea comprises the following steps:
1) inoculating the detection bacteria to a Columbia blood plate, and culturing the inoculated plate in a constant temperature box at 35-37 ℃ for 48 h;
2) selecting a single colony on a plate, coating a target plate, adding a matrix solution immediately after a mycoderm is dried in the air, and detecting by using MALDI-TOF MS after the mycoderm is dried in the air; comparing the obtained detected bacteria map with the map in the map database of the invention, and judging the bacteria map to be positive when the Log (score) is more than or equal to 2.0.
The matrix solution is a 14mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving a basic solvent; the base solvent contained acetonitrile at a final volume concentration of 50%, trifluoroacetic acid at a final volume concentration of 2.5%, and the balance water.
The method 2 comprises the following steps:
the method for rapidly identifying Burkholderia melioidea comprises the following steps:
1) inoculating the detected bacteria on a chocolate flat plate, and culturing the inoculated flat plate in a constant temperature box at 35-37 ℃ for 24 hours;
2) selecting a single colony on a plate, coating a target plate, adding a matrix solution immediately after a mycoderm is dried in the air, and detecting by using MALDI-TOF MS after the mycoderm is dried in the air; comparing the obtained detected bacteria map with the map in the map database of the invention, and judging the bacteria map to be positive when the Log (score) is more than or equal to 2.0.
The matrix solution is a 14mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving a basic solvent; the base solvent contained acetonitrile at a final volume concentration of 50%, trifluoroacetic acid at a final volume concentration of 2.5%, and the balance water.
The method 3 comprises the following steps: the method for rapidly identifying Burkholderia melioidea comprises the following steps:
1) inoculating the detected bacteria to a Macconkey plate, and culturing the inoculated plate in a thermostat at 35-37 ℃ for 48 hours;
2) selecting a single colony on a plate, coating a target plate, adding a matrix solution immediately after a mycoderm is dried in the air, and detecting by using MALDI-TOF MS after the mycoderm is dried in the air; comparing the obtained detected bacteria map with the map in the map database of the invention, and judging the bacteria map to be positive when the Log (score) is more than or equal to 2.0.
The matrix solution is a 14mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving a basic solvent; the base solvent contained acetonitrile at a final volume concentration of 50%, trifluoroacetic acid at a final volume concentration of 2.5%, and the balance water.
The clinically collected samples were identified by methods 1-3 as described above and confirmed by 16S rRNA sequencing, showing: method 3 failed to obtain all the 38 characteristic peak data provided in the foregoing example 1, and the identification showed an accuracy of 72.5% and methods 1 and 2 identified an accuracy of 99.9%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A burkholderia farci identification map database is characterized in that the database comprises a protein map with the molecular weight of 2000-20000 of burkholderia farci, and the protein map is formed by drawing the mass-to-mass ratio and the relative strength of proteins with different molecular weights;
the protein with the molecular weight of 2000-3000 has 3 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 2190.8179, 2697.1714, 2840.8544;
the protein with the molecular weight of 3000-4000 has 9 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 3132.8089, 3210.5085, 3144.2096, 3210.5085, 3278.7055, 3583.1600, 3640.8447, 3856.7721, 3938.5076;
the protein with the molecular weight of 4000-6000 has 7 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 4366.7200, 4451.0223, 4769.6272, 4778.9532, 5070.4933, 5348.3594, 5381.5558;
the protein with the molecular weight of 6000-8000 has 7 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 6255.3870, 6316.1295, 6387.0116, 7158.8015, 7274.6620, 7708.2632, 7872.5290;
the protein with the molecular weight of 8000-10000 has 6 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 8328.4155, 8880.0214, 9067.7875, 9230.8977, 9560.6676, 9746.2004;
the protein with the molecular weight of 10000-12000 has 4 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 10145.7753, 10308.1668, 10704.8187, 11234.7706;
the protein with the molecular weight of 12000-14000 has 2 characteristic peaks, and the mass-to-charge ratios are respectively as follows: 12224.8512, 13026.6093.
2. The method for rapidly identifying Burkholderia melioidea is characterized by comprising the following steps of:
1) inoculating the detected bacteria to a flat plate, and culturing the inoculated flat plate in a constant temperature box at 35-37 ℃ for 20-48 h;
2) selecting a single colony on a plate, coating a target plate, adding a matrix solution immediately after a mycoderm is dried in the air, and detecting by using MALDI-TOFMS after the mycoderm is dried in the air; comparing the obtained map with the map in the database of claim 1, and judging the map to be positive when Log (score) is more than or equal to 2.0.
3. The method for rapidly identifying Burkholderia melioidis according to claim 2, wherein the matrix solution is a 12-15 mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving a basic solvent.
4. The method for rapidly identifying Burkholderia melioidis according to claim 3, wherein the base solvent comprises acetonitrile at a final volume concentration of 45-55%, trifluoroacetic acid at a final volume concentration of 2.3-2.8%, and the balance of water.
5. The method for rapidly identifying Burkholderia melioidis according to claim 2, wherein the matrix solution is a 14mg/L alpha-cyano-4-hydroxy-cinnamic acid solution prepared by dissolving in a basic solvent; the base solvent contained acetonitrile at a final volume concentration of 50%, trifluoroacetic acid at a final volume concentration of 2.5%, and the balance water.
6. The method for rapidly identifying Burkholderia melioidis according to claim 2, wherein the plate is a Columbia blood plate or a chocolate plate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105548338A (en) * | 2015-12-10 | 2016-05-04 | 山东出入境检验检疫局检验检疫技术中心 | Protein fingerprint model for Burkholderia gladioli and application thereof |
| US20200104464A1 (en) * | 2018-09-30 | 2020-04-02 | International Business Machines Corporation | A k-mer database for organism identification |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548338A (en) * | 2015-12-10 | 2016-05-04 | 山东出入境检验检疫局检验检疫技术中心 | Protein fingerprint model for Burkholderia gladioli and application thereof |
| US20200104464A1 (en) * | 2018-09-30 | 2020-04-02 | International Business Machines Corporation | A k-mer database for organism identification |
Non-Patent Citations (2)
| Title |
|---|
| JIN LI等: ""Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identifying Burkholderia pseudomallei and Burkholderia thailandensis isolates"", 《EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES》 * |
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Application publication date: 20210910 |