CN114354946A - Method for establishing regional human pathogenic bacteria polypeptide quality reference spectrum library - Google Patents
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Abstract
The invention relates to a method for establishing a regional human pathogenic bacteria polypeptide quality reference spectrum library. The method for establishing the regional human pathogenic bacteria polypeptide quality reference spectrum library comprises the following steps: (1) collecting local common human pathogenic bacteria; (2) collecting the human pathogenic bacteria to carry out protein fingerprint spectrum; (3) analyzing the protein fingerprint of the human pathogenic bacteria, and establishing a regional characteristic spectrum database of the human pathogenic bacteria after collecting characteristic spectrums, namely the human pathogenic bacteria polypeptide quality reference spectrum database. According to the method for establishing the human pathogenic bacteria polypeptide quality reference spectrum library, the constructed local PMRSL databases of staphylococcus aureus, escherichia coli and acinetobacter baumannii meet the requirement of mass spectrum detection and identification, can be used for identifying the local human pathogenic bacteria staphylococcus aureus, escherichia coli and acinetobacter baumannii, and meanwhile, the original reference database is expanded, and the accuracy and the integrity of database identification are improved.
Description
Technical Field
The invention particularly relates to a method for establishing a regional human pathogenic bacteria polypeptide quality reference spectrum library.
Background
The occurrence and development of pathogenic bacteria infection form a serious threat to human health, the treatment of drug-resistant bacteria poses a serious challenge to human life, northwest of our district is a place where multiple nations inhabit, and the infection and epidemic characteristics of pathogenic bacteria and the like have certain differences from the inland.
The matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) technology has the advantages of rapidness, accuracy and low cost for species identification of microorganisms, and the technology is used for carrying out cluster analysis on a polypeptide mass spectrum of an unknown sample acquired by an instrument and spectra of known strains in a database to obtain an identification result. Therefore, the accuracy and specificity of species identification depend on the integrity and reliability of the MALDI-TOF MS spectrum database of the microorganism, which is prepared by the manufacturer or user of MALDI-TOF MS, i.e., the database is the core of MALDI-TOF MS identification of the microorganism. At present, research aiming at applying a large number of pathogenic microorganisms in a certain country or region to system analysis is still lacked, namely, identification databases of different pathogenic bacteria in different regions are not established in China.
In view of the above, the invention provides a method for establishing a regional human pathogenic bacteria polypeptide quality reference spectrum library, which can be used for quickly and accurately identifying local human pathogenic bacteria staphylococcus aureus, escherichia coli and acinetobacter baumannii.
Disclosure of Invention
The invention aims to provide a method for establishing a human pathogenic bacteria polypeptide quality Reference spectrum library, a constructed local PMRSL Database of staphylococcus aureus, escherichia coli and acinetobacter baumannii meets the requirement of mass spectrum detection and identification, can be used for identifying local common pathogenic bacteria staphylococcus aureus, escherichia coli and acinetobacter baumannii, expands an Original Reference Database (ORD) and improves the accuracy and the integrity of Database identification.
In order to realize the purpose, the adopted technical scheme is as follows:
the method for establishing the regional human pathogenic bacteria polypeptide quality reference spectrum library comprises the following steps:
(1) collecting local common human pathogenic bacteria;
(2) collecting the human pathogenic bacteria to carry out protein fingerprint spectrum;
(3) analyzing the protein fingerprint of the human pathogenic bacteria, and establishing a regional characteristic spectrum database of the human pathogenic bacteria after collecting characteristic spectrums, namely the human pathogenic bacteria polypeptide quality reference spectrum database.
Further, in the step (1), the human pathogenic bacteria screened by clinical separation are identified by 16S rRNA sequencing.
Furthermore, in the step (1), the common pathogenic bacteria of human origin are staphylococcus aureus, escherichia coli and acinetobacter baumannii.
Further, in the step (3), the characteristic data of the protein fingerprint of the human pathogenic bacteria is analyzed through a MicroID detection system.
Further, the establishing method also comprises a step (4) of verifying the standard strain and a large amount of human homologous pathogenic bacteria.
Compared with the prior art, the invention has the beneficial effects that:
the MicroID detection system is a Xinhui microorganism detection and identification system, namely a microorganism detection cloud computing platform, and is a common characteristic spectrum formed by characteristic spectrums of more than 3000 species, more than 60000 strains and multi-pearl bacteria in total by the China center for disease prevention and control (CDC). The source of the strain is in the Chinese range, the representativeness of Chinese native strains is fully embodied, and the species and the range of the strain map are wide. Because of the differences of pathogenic bacteria in different regions, the MALDI-TOF MS technology still lacks a detection system or identification database for a large number of pathogenic microorganisms in a certain region.
The invention adopts MALDI-TOF MS technology, and screens common pathogenic bacteria from clinical separation by 16S rRNA sequencing: staphylococcus aureus, Escherichia coli and Acinetobacter baumannii, then protein fingerprint spectrum collection is carried out on the screened pathogenic bacteria, a MicroID detection system is introduced, a core algorithm and a computer processing system are utilized to analyze the characteristic data of the protein fingerprint spectrum of the pathogenic bacteria, the characteristic spectrum of the pathogenic bacteria is collected, a local clinical common pathogenic bacteria Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii regional characteristic spectrum library, namely a Polypeptide Mass Reference Spectrum Library (PMRSL), is established, and the standard strains and a large amount of human same pathogenic bacteria are used for evaluating the local pathogenic bacteria, so that the accuracy and the practicability of a newly-established local PMRSL database are verified, and the richness and the integrity of the local PMRSL database are increased.
Drawings
FIG. 1 shows the electrophoresis results of PCR products of some strains.
Detailed Description
In order to further illustrate the method for establishing the regional reference spectrum library for the polypeptide of the human pathogenic bacteria to achieve the intended purpose of the invention, the following detailed description is provided for the method for establishing the reference spectrum library for the polypeptide of the human pathogenic bacteria according to the present invention with reference to the preferred embodiments, and the specific implementation, structure, characteristics and efficacy thereof. In the following description, different "one embodiment" or "an embodiment" refers to not necessarily the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The method for establishing the reference spectrum library of the polypeptide quality of pathogenic bacteria of the inventor is further described in detail by combining specific examples as follows:
the technical scheme of the invention is as follows:
the method for establishing the regional human pathogenic bacteria polypeptide quality reference spectrum library comprises the following steps:
(1) collecting local common human pathogenic bacteria;
(2) collecting the human pathogenic bacteria to carry out protein fingerprint spectrum;
(3) analyzing the protein fingerprint of the human pathogenic bacteria, and establishing a regional characteristic spectrum database of the human pathogenic bacteria after collecting characteristic spectrums, namely a polypeptide mass reference spectrum database (PMRSL) of the human pathogenic bacteria.
Preferably, in the step (1), common human pathogens are selected by identifying and clinically separating 16S rRNA sequencing.
Preferably, in the step (1), the common pathogenic bacteria of human origin are staphylococcus aureus, escherichia coli and acinetobacter baumannii.
Preferably, in the step (3), the characteristic data of the protein fingerprint of the human pathogenic bacteria is analyzed by a micro id detection system.
Preferably, the establishing method further comprises the step (4) of verifying the standard strain and a large number of human homologous pathogenic bacteria.
Examples
Screening and molecular identification of human pathogenic bacteria
Based on the infection rate, the drug resistance rate, the detection rate and the like of pathogenic bacteria in a CHINET Chinese bacterial drug resistance detection report, the selected strains are human pathogenic bacteria isolates obtained from different people in different years and different sources. All strains were identified by molecular and biochemical analysis, cultured according to the standard methods of CLSI (clinical laboratory standards institute), and screened for the identified strains, i.e., Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, total 300 beads, as follows:
1. materials and reagents
TABLE 1 test Main materials and reagents
2. Apparatus and device
TABLE 2 Main apparatus and equipment for testing
3. Pathogenic bacteria screening and preservation
3.1 the strain source: the total 345 strains of staphylococcus aureus, escherichia coli and acinetobacter baumannii which are separated from a fifth subsidiary hospital clinical laboratory of Xinjiang medical university in 1 month to 2020 in 2019.
3.2 identification and selection of pathogenic bacteria: the preliminary identification of staphylococcus aureus, escherichia coli and acinetobacter baumannii is carried out by a merriella full-automatic bacteria identifier VITEK 2-Compact.
3.3 culture and cryopreservation of pathogenic bacteria: the culture method and conditions of the strain are carried out according to CLSI standard specifications, all pathogenic bacteria adopt blood agar nutrient medium, streak culture is carried out at 37 ℃, single colony is repeatedly picked and streaked for 2-3 times, and pure cultured single colony is obtained.
3.4 cryopreservation of pathogenic bacteria: picking single colony, transferring the single colony to a nutrient agar plate, performing streak culture, and culturing at 37 ℃ for 24 h; the next day, scraping the colony, placing into a freezing tube, adding equal amount of nutrient broth and sterilized 30% glycerol, mixing, cooling at-20 deg.C and-80 deg.C, and storing at-80 deg.C.
4. Analytical identification of pathogenic bacteria
4.1 extraction of DNA from pathogenic bacteria
Taking 2mL of bacterial liquid, centrifuging at 1000rpm for 30s, and removing supernatant and precipitating; adding 1mL of CTAB (2%) lysis solution, mixing uniformly, adding 20 mu L of proteinase K, and mixing uniformly by vortex oscillation; bathing at 56 deg.C for 1-2 h; mixing by reversing every 20 min; adding 350 mu L of phenol and 350 mu L of chloroform, and fully and uniformly mixing; standing for 3 min; centrifuging at 12000r/min for 12 min; transferring the supernatant to a new 2mL centrifuge tube; adding 1.2mL of absolute ethyl alcohol, and evenly mixing for 20 times by reversing; standing at-20 deg.C for 30 min; centrifuging at room temperature of 10000r/min for 10 min; pouring off the supernatant, adding 700 μ L70% anhydrous ethanol, mixing by inversion, and standing at room temperature for 3 min; centrifuging at 10000r/min for 5 min; pouring off the supernatant, and centrifuging at 10000r/min for 1 min; sucking off residual liquid at the bottom of the pipe, uncovering and airing for 5-10 min; adding 60-100 μ L of Tris-Cl (pH8.0-8.3)10 mM; dissolving at 65 deg.C for 1 h. 5-time dilution: mu.L Tris and 2. mu.L stock solution. Electrophoresis: 0.8% agarose gel, 2. mu.L of load.
4.2PCR amplification
4.2.1 primer design
Based on the gene sequence information, universal primers were designed and synthesized using Primer 5 software, and the Primer sequences are shown in Table 3.
TABLE 3 primer sequences
4.2.2PCR amplification conditions
Adopting a 25 mu L reaction system of a Big-dye kit, pre-denaturing at 95 ℃, 300s, amplifying for 35 cycles, and circulating parameters: the temperature was maintained at 95 ℃ for 30 seconds, 58 ℃ for 30 seconds, 72 ℃ for 60 seconds, and then at 72 ℃ for 300 seconds. The PCR reaction system is shown in Table 4.
TABLE 4PCR reaction System
4.3 electrophoretic test
And (3) performing gel electrophoresis on the amplified product, selecting an electrophoresis detection result with clear band and no degradation, and performing subsequent experiments on the sample. Partial product results are shown in figure 1.
4.416S rRNA sequencing identification
The sequencing procedure was performed according to the Applied Biosystems Sanger kit instructions:
4.4.1 recovering agarose gel containing a target band, and adding a gel block melting solution Binding Buffer into the gel block; melting the rubber blocks at 56 deg.C after uniform mixing, and mixing for 7min under intermittent oscillation to melt the rubber blocks;
4.4.2 Place Spin Column in the kit on the Collection Tube; transferring the solution obtained in the operation step 1 into a Spin Column, centrifuging for 1min at 8000r/min, and removing the filtrate;
4.4.3 adding 700 μ L of Wash Buffer into Spin Column, room temperature 8000r/min, centrifuging for 1min, and discarding the filtrate; repeating the above steps;
4.4.4 Spin Column was mounted on the Collection Tube at 12000r/min, centrifuged for 2 min; placing Spin Column on a new 1.5mL centrifuge tube, adding 25 μ L of precipitation Buffer at the center of the Spin Column membrane, standing at room temperature for 1min, and centrifuging at 8000r/min for 1min to elute DNA; sequencing the recovered product by using forward and reverse primers, and splicing the sequencing result.
Carrying out 16S rRNA sequencing on 345 strains of 102 collected staphylococcus aureus, 142 escherichia coli and 101 acinetobacter baumannii, comparing through an NCBI database, identifying the species level, screening out qualified strain samples according to the identification result and the sample preservation quality, and carrying out protein fingerprint spectrum collection. The 16S rRNA sequencing results were: through molecular identification, 2 strains of staphylococcus aureus are not amplified, 3 strains are identified as non-staphylococcus aureus, namely 97 beads are qualified strains; 5 E.coli strains are not amplified, 8 E.coli strains are identified as non-E.coli strains, namely 129 bead is a qualified strain; acinetobacter baumannii strain 1 is not amplified, and 17 strains are identified as non-Acinetobacter baumannii, namely 83 beads are qualified strains.
Second, MALDI-TOF MS identification of humanized pathogenic bacteria staphylococcus aureus, Escherichia coli and Acinetobacter baumannii in Ullus city of Sinkiang and establishment of polypeptide mass reference spectrum library
The method comprises the steps of collecting protein fingerprints of staphylococcus aureus, escherichia coli and acinetobacter baumannii, introducing a core leading detection system-a MicroID microorganism detection and identification system, and constructing a Wulu wood city clinical pathogenic bacteria regional characteristic spectrum database by means of a unique core algorithm and a computer processing system by means of MALDI-TOF MS technology. The method comprises the steps of bringing in the map of variant and characteristic pathogenic bacteria, and innovatively constructing a rapid identification and detection platform for pathogenic bacteria in regions of Wuluqiqi city.
1. Materials and reagents
Relevant materials and reagents are shown in table 5; escherichia coli, Staphylococcus aureus, Acinetobacter baumannii standard strains were all from the CDC strain database.
TABLE 5 test Main materials and reagents
2. Apparatus and device
The relevant instruments and equipment are shown in Table 6.
TABLE 6 main apparatus and equipment for testing
MALDI-TOF MS spectrum acquisition
3.1 sample preparation
About 5mg of the colony was picked up by an inoculating loop, placed in a 1.5mL centrifuge tube containing 300. mu.L of pure water, and shaken well. Adding 900 mu L of absolute ethyl alcohol into the centrifuge tube, shaking, and uniformly mixing for 3-5 s by using a vortex instrument. Mixing, centrifuging at 12000r/min for 2min, discarding supernatant, centrifuging at 12000r/min for 2min, removing residual liquid, and drying at room temperature for 5 min. Adding 40 mu L of 70% formic acid, fully shaking, uniformly mixing for 3-5 s by using a vortex instrument, then adding 40 mu L of acetonitrile, fully shaking, uniformly mixing for 3-5 s by using the vortex instrument, centrifuging for 2min at 12000r/min by using a centrifugal machine, sucking 1 mu L of supernatant by using a liquid transfer gun, counting on a target plate, adding 1 mu L of matrix solution to cover a sample spot after drying at room temperature, and naturally drying at room temperature.
3.2 preparation of the matrix solution
10mg of CHCA was added to 1mL of 50% ACN-2.5% TFA in water.
3.3 data acquisition and acquisition
Three persons operated the same batch of samples and extracted protein samples respectively. During identification, each person of the same strain respectively points 24 points of the target to collect the atlas, and three persons collect 72 atlases in total. Preprocessing each strain data through a peak searching algorithm, solving the problems of baseline drift, obvious noise and the like existing in the original data, finding out obvious signal expression points, namely characteristic peaks, and selecting 70 strong characteristic maps for clustering analysis.
3.4MALDI-TOF MS Collection of bacterial peptide Mass Spectroscopy parameters
The laser energy is 64%, the number of spectrograms of each target point is 100, the bombardment number of each spectrogram is 5, and a linear positive ion mode is selected. The quality of the characteristic peak data of the sample is evaluated by cluster analysis, which reflects the mass spectrum difference among a plurality of spectrograms of the same strain, the proportion is between 0 and 100 percent, and the lower the percentage is, the larger the spectrogram difference is. More diverse data can be excluded from the cluster analysis plot when creating the peptide mass reference profile. Spectrogram with similarity over 70% is selected for clustering analysis.
4. Construction of a database
And constructing a peptide mass reference spectrum by using a local library building function of the MicroID. The parameters are set as follows: the mass range is 3000-15000; maximum mass error: 600 PPM; minimum matching rate: 70 percent; maximum peak number: 70. and each warehousing reference spectrum is a spectrogram formed by normalizing, smoothing, base line correcting and peak selecting 24 in 3 batches and 24 in each batch, and a remarkable common peak series is obtained by a clustering analysis algorithm.
5. Database validation and evaluation
Cross-validation methods, i.e. performing a database search to exclude reference spectra of strains to be identified, were used in the study. The species comprising only one strain need not be verified. Determining the recognition degree according to the interpretation standard of the MicroID system: the total score is 10 points, less than 6 points are not recognizable, 6 points to 8 points are referable, 8 points to 9.2 points are credible (belonging to the identification of the level), and more than 9.2 points are highly credible (identification of the seed level). The local libraries were searched using 100 strains each of Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii and their standard strains identified by 16s rRNA for validation to evaluate the confidence of this time the new reference database established by the MicroID system in the identification at the genus and species levels.
6. Statistical analysis
Comparing and analyzing the 16S rRNA gene sequencing result by using an NCBI database; clustering analysis of MALDI-TOF MS peptide mass reference spectra was performed using MicroID version 4.0 analysis. Verification of the local library was performed using Shimadzo Biotech MALDI-MS 2.9.3.
7 results
7.1 identification result and library construction verification of Staphylococcus aureus
7.1.1MALDI-TOF MS Spectroscopy screening
Collecting protein mass spectrum data by a MALDI-TOF MS method, screening the obtained data by clustering analysis, preserving maps with similarity of more than 70%, removing samples which are not staphylococcus in 16S rRNA sequencing, and finally screening 29 strains of staphylococcus aureus.
7.1.2 MALDI-TOF MS identification result of Staphylococcus aureus
And performing cluster analysis on the acquired staphylococcus aureus protein fingerprint to construct a staphylococcus aureus spectrogram. And carrying out normalization treatment on the spectra with the similarity of more than 70 percent to obtain the protein fingerprint characteristic spectrum of the corresponding strain.
The staphylococcus aureus spectra collected at this time have eight characteristic peaks at m/z3211, m/z3445, m/z4305, m/z4815, m/z5033, m/z5549, m/z6892 and m/z9641 after alignment. Comparing the characteristic peaks obtained by the analysis and comparison after the normalization with the characteristic peaks in the existing bacteria library, and obtaining the result shown in table 7, wherein m/z5549 and m/z9641 have fewer similar peaks in the standard bacteria library, namely the characteristic peaks are considered to be regional characteristic peaks of staphylococcus aureus.
TABLE 7 MALDI-TOF MS spectrum comparison of Staphylococcus aureus
7.1.3 verification of data authentication capabilities
The sample profiles of 29 S.aureus strains selected at this time were collected and pooled, and the database created at this time was searched for the results of identification using 100 strains of 69 S.aureus strains identified using 16s rRNA and 31 standard strains selected from the CDC library. The identification results of 100 strains of bacteria in the self-built library are high in credibility of 99 percent and credibility of 1 percent, and are shown in Table 8. The high credibility of the identification result is more than 90%, so that the 29 screened staphylococcus aureus meets the mass spectrum detection requirement and can be used for establishing a database.
TABLE 8 Staphylococcus aureus Standard bacteria local library identification results
7.2 identification and library construction verification results of Escherichia coli
7.2.1 MALDI-TOF MS Spectroscopy screening
Collecting protein mass spectrum data by a MALDI-TOF MS method, screening the obtained data by cluster analysis, preserving maps with similarity of more than 70%, removing samples which are not escherichia coli by 16S rRNA sequencing, and finally screening 42 escherichia coli strains.
7.2.2 MALDI-TOF MS identification
And performing cluster analysis on the collected Escherichia coli protein spectrogram to construct a stereo Escherichia coli spectrogram. And (4) carrying out normalization treatment on 897 spectra with the similarity of more than 70% to obtain the protein characteristic spectrum of the corresponding strain.
As can be seen from the post-alignment, the Staphylococcus aureus pattern collected this time has characteristic peaks at m/z3131, m/z4166, m/z4364, m/z4535, m/z4775, m/z5096, m/z5382, m/z5751, m/z6257, m/z6317, m/z 9550. Comparing the characteristic peak analyzed and compared after normalization with the characteristic peak in the existing bacteria library to obtain the peak analyzed this time, and the result is shown in Table 9. Of these, m/z4166 and m/z4364 have fewer similar peaks in the standard bacterial library.
TABLE 9 MALDI-TOF MS spectrum comparison of Escherichia coli
7.2.3 database identification capability verification
A total of 42 Escherichia coli sample maps were collected and pooled, and 100 Escherichia coli 88 identified by the remaining 16s rRNA and 12 Escherichia coli standard strains were used to search and identify the results in the database created this time. The identification results of 100 strains of bacteria in the self-built library are high in credibility of 96 percent and credibility of 4 percent, and are shown in table 10. The high credibility of the identification result is more than 90%, so that the 42 screened escherichia coli strains meet the mass spectrum detection requirement and the database can be established.
TABLE 10 Escherichia coli Standard bacteria local library identification results
7.3 identification and library construction verification results of Acinetobacter baumannii
7.3.1 MALDI-TOF MS Spectroscopy screening
Collecting protein mass spectrum data by MALDI-TOF MS method, screening the obtained data by cluster analysis, preserving the map with similarity of more than 70%, removing the sample which is not Acinetobacter baumannii in 16S rRNA sequencing, and finally screening 19 Acinetobacter baumannii strains
7.3.2 Acinetobacter baumannii Strain MALDI-TOF MS identification result
And performing cluster analysis on the collected acinetobacter baumannii protein spectrogram to construct an acinetobacter baumannii spectrogram. And (3) performing normalization treatment on 902 spectra with the similarity of more than 70% to obtain the protein characteristic spectrum of the corresponding strain.
After post-alignment, it can be seen that the Acinetobacter baumannii maps have characteristic peaks at m/z4247, m/z5175, m/z5432, m/z5750 and m/z 8495. The characteristic peak analyzed and compared after normalization is compared with the characteristic peak in the existing bacteria library to obtain the peak analyzed this time, and the result is shown in table 20.
TABLE 11 MALDI-TOF MS spectrum alignment of Acinetobacter baumannii
7.3.3 database identification capability verification
Collecting and summarizing 19 Acinetobacter baumannii sample maps for library construction, using 100 strains of 64 Acinetobacter baumannii subjected to 16S rRNA and 16 Acinetobacter baumannii standard strains, and searching and identifying the database built at this time to obtain results. The identification results of 100 strains of bacteria to be detected in the self-built library are high in credibility of 62 percent and credibility of 38 percent, and are shown in Table 12. The high credibility of the identification result is more than 60%, and the credibility is more than 100%, so that the 29 screened Acinetobacter baumannii meet the mass spectrum detection requirements, and the database can be established.
TABLE 12 identification results of Acinetobacter baumannii standard bacteria local library
Three, result in
Protein maps of Escherichia coli (42 strains), staphylococcus aureus (29 strains) and Acinetobacter baumannii (19 strains) screened by 16S rRNA sequencing identification are collected, after the protein maps of three pathogen samples are subjected to cluster analysis, the proportion of the maps with the similarity of more than 70% is more than 87%, and the three pathogen samples are high in similarity, can be identified as the same species and are consistent with the 16S rRNA sequencing identification result. The protein maps with the similarity of the three pathogenic bacteria of more than 70 percent are subjected to collective treatment to obtain representative characteristic peaks, wherein 8 staphylococcus aureus (m/z3211, m/z3445, m/z4305, m/z4815, m/z5033, m/z5549, m/z6892 and m/z9641) have fewer similar peaks in a standard bacteria library; escherichia coli has 11 species (m/z3131, m/z4166, m/z4364, m/z4535, m/z4775, m/z5096, m/z5382, m/z5751, m/z6257, m/z6317, m/z9550), wherein m/z4166, m/z4364 have fewer similar peaks in the standard pool; there are 5 species of Acinetobacter baumannii (m/z4247, m/z5175, m/z5432, m/z5750, m/z 8495). Establishing a local library through characteristic peaks and protein map data, and performing retrieval and identification in the local library by using 300 strains of staphylococcus aureus, escherichia coli, acinetobacter baumannii and standard strains thereof, wherein the result shows that 99 percent of identification results of 100 standard strains of staphylococcus aureus in the local library are highly credible, and 1 percent of identification results are credible; the identification results of 100 escherichia coli standard strains in the local library are high in credibility of 96% and credibility of 4%; the identification result of 100 Acinetobacter baumannii standard strains in the local library is high in credibility of 62 percent and credibility of 38 percent. The high credibility of the screened Escherichia coli and Staphylococcus aureus is above 90%, and the credibility is above 100%; the high credibility of the acinetobacter baumannii is more than 60%. Therefore, the 42 Escherichia coli strains, 29 staphylococcus aureus strains and 19 acinetobacter baumannii strains constructed at this time all meet the requirement of mass spectrometry detection and identification, and can be used as verification strains to expand a polypeptide quality reference spectrum library (PMRS). Most of the bacteria in the standard bacteria library come from Beijing, Guangdong and the like, and the high credibility of the identification result is greatly improved after the characteristic peaks detected by the staphylococcus aureus and the Escherichia coli are expanded to the reference spectrum library.
Under the influence of climate, environment and economic factors of different regions, mass spectrum characteristic peaks have certain differences, so that certain strains fail to be identified. In order to realize the best matching and improve the typing and identifying capability, enough pathogenic bacteria are required to be used as a characteristic map of a local bacterial strain of a known strain as a database, and with the extensive research on biological labels and the continuous improvement of a database of the map library, the MAL-DI-TOF MS is more and more widely applied. By constructing the PMRS and evaluating the PMRS by using a large number of human pathogens, the original reference database is expanded, and the identification accuracy and specificity for human pathogenic microorganisms are improved. The PMRS database is expected to be developed and used in a plurality of fields such as clinical diagnosis and treatment, disease control, quality assurance, food safety monitoring and the like.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (5)
1. The method for establishing the regional human pathogenic bacteria polypeptide quality reference spectrum library is characterized by comprising the following steps of:
(1) collecting local common human pathogenic bacteria;
(2) collecting the human pathogenic bacteria to carry out protein fingerprint spectrum;
(3) analyzing the protein fingerprint of the human pathogenic bacteria, and establishing a regional characteristic spectrum database of the human pathogenic bacteria after collecting characteristic spectrums, namely the human pathogenic bacteria polypeptide quality reference spectrum database.
2. The method of establishing according to claim 1,
in the step (1), the human pathogenic bacteria clinically separated and screened are identified by 16S rRNA sequencing.
3. The method of establishing according to claim 1,
in the step (1), common human pathogenic bacteria are staphylococcus aureus, escherichia coli and acinetobacter baumannii.
4. The method of establishing according to claim 1,
and (3) analyzing the characteristic data of the protein fingerprint of the human pathogenic bacteria by a MicroID detection system.
5. The method of establishing according to claim 1,
the establishing method also comprises a step (4) of verifying the standard strain and a large amount of human homologous pathogenic bacteria.
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