CN110501414B - Identification model, construction method and application of VIM type and SPM type metalloenzyme pseudomonas aeruginosa - Google Patents
Identification model, construction method and application of VIM type and SPM type metalloenzyme pseudomonas aeruginosa Download PDFInfo
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Abstract
The invention relates to a VIM type and SPM type metalloenzyme pseudomonas aeruginosa identification model, a construction method and application, wherein MALDI-TOF MS is used for analyzing mass spectrum protein characteristic peaks of the VIM type and SPM type metalloenzyme pseudomonas aeruginosa, and establishing an identification model for distinguishing two subtypes; the identification model can quickly and accurately identify the VIM type and SPM type metalloenzyme pseudomonas aeruginosa, and has the advantages of simple and convenient operation, short time consumption and the like, so a brand new thought is provided for the research of identifying the bacterial subtype by MALDI-TOF MS, and the identification model has a very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of clinical microbiological examination, and relates to a VIM (virginia model) and SPM (SPM) metalloenzyme pseudomonas aeruginosa identification model, a construction method and application.
Background
MALDI-TOF MS is a new kind of microorganism identification technology that developed rapidly in recent years, have been applied to clinical microorganism laboratory gradually at present, it can be through comparing the difference of mass spectrum peaks such as conservative protein such as bacterium specificity ribosomal protein, nucleic acid binding protein, heat shock protein, etc. and other housekeeping gene protein with higher content, can distinguish different subtypes of pathogenic bacterium, have easy and simple to handle, detect advantages such as being fast and accurate, high-throughput; however, the VITEK MS database introduced by the French organism Merrieia cannot identify the metalloenzyme Pseudomonas aeruginosa subtype.
At present, the metal enzyme phenotype preliminary screening test is mainly carried out in a laboratory by using an imipenem EDTA double-paper synergistic experiment, and the parting identification can be carried out on VIM type and SPM type genes of pseudomonas aeruginosa producing metal enzyme through metalloenzyme gene PCR amplification and sequence analysis. Based on the research of molecular epidemiology, the epidemic situation and the change trend of the Pseudomonas aeruginosa, which is the metalloenzyme VIM and SPM, can be determined, and scientific basis is provided for the research of the drug resistance mechanism of the Pseudomonas aeruginosa and the infection control. However, the method has the defects of long time consumption, complex operation, easy pollution, inapplicability to clinical rapid detection and the like. Therefore, a new method for rapidly and accurately detecting Pseudomonas aeruginosa, which is a VIM-type metalloenzyme and a SPM-type metalloenzyme, is urgently needed to be researched.
Disclosure of Invention
In view of the above, the present invention aims to provide a model for identifying pseudomonas aeruginosa, which is a VIM-type and SPM-type metalloenzyme, a construction method and an application thereof.
1. A construction method of a VIM type and SPM type metalloenzyme pseudomonas aeruginosa identification model comprises the following specific steps:
(1) firstly, introducing mass spectrum results of VIM-type and SPM-type strains into an SARAMIS database, and finding out partial different mass spectrum peaks between the VIM-type and SPM-type strains by clustering analysis of mass spectrum peaks of the VIM-type and SPM-type strains; then, the two mass spectrum peaks are compared and analyzed through LaunchPad software to obtain 17 high signal intensity peaks serving as the common mass peaks of the two;
(2) in order to screen characteristic peaks which are different between VIM type strains and SPM type strains, the launchPad software is used again to respectively compare the mass spectrum characteristic peaks of the VIM type strains and the non-VIM type strains as well as the mass spectrum characteristic peaks of the SPM type strains and the non-SPM type strains;
(3) and finally, selecting 4 characteristic peaks of the VIM type strain and 3 characteristic peaks of the non-VIM type strain as models for identifying the VIM type, and selecting 2 characteristic peaks of the SPM type strain and 2 characteristic peaks of the non-SPM type strain as models for identifying the SPM type.
Preferably, in step (1), the screening method for the strains of VIM type and SPM type is as follows: firstly, carrying out a phenotype preliminary screening experiment on 258 imipenem drug-resistant pseudomonas aeruginosa to obtain 106 MBL phenotype positive strains, and 152 pseudomonas aeruginosa not producing metalloenzyme; then respectively carrying out PCR amplification on IMP, VIM, SPM, GIM, SIM and NDM genes by taking 106 MBL phenotype positive strain DNAs as templates; and finally, connecting the MBL gene positive PCR product to a TA carrier, and sequencing and confirming the connecting carrier to obtain VIM type positive 40 strains and SPM type positive 35 strains without amplifying IMP, SIM, GIM and NDM positive strains.
Preferably, in the step (1), the cluster analysis is performed according to a method described by mass spectrum identification of VITEK MS RUO and Standard Operation Process (SOP) of a scientific research instrument, mass spectrum results of VIM-type and SPM-type strains are led into an SARAMIS database, and then cluster analysis is performed on the number of peaks with the same mass of the two strains through SARAMIS software, so that the whole detection and analysis time only takes a few minutes, and the processing mode is simple and rapid.
Preferably, in the step (1), 15 strains of VIM type and 15 strains of SPM type are randomly selected and subjected to cluster analysis based on the number of peaks having the same mass.
Preferably, in step (1), the 17 high signal intensity peaks comprise: 3601m/z,4434m/z,4544m/z,4849m/z,4992m/z,5114m/z,5210m/z,5737m/z,6046m/z,6348m/z,6676m/z,7203m/z,7595m/z,8786m/z,9089m/z,9987m/z and 10229 m/z.
Preferably, in step (2), the non-SPM forms include SPM forms and pseudomonas aeruginosa non-metallogenic enzymes.
Preferably, in step (3), the 4 characteristic peaks of the VIM-type strain are: 3230m/z,3409m/z,5450m/z,7329m/z, and 3 characteristic peaks of non-VIM strains are 3429m/z,5490m/z, and 7390 m/z.
Preferably, in step (3), 2 characteristic peaks of the SPM-type strain are 3663m/z and 6264m/z, and 2 characteristic peaks of the non-SPM-type strain are 4068m/z and 4282 m/z.
2. The identification model of the Pseudomonas aeruginosa with the VIM type and SPM type metalloenzymes is constructed by the method.
3. The application of the recognition model in the identification of Pseudomonas aeruginosa through VIM type and SPM type metalloenzymes.
4. A method for quickly and accurately identifying VIM and SPM metalloenzyme pseudomonas aeruginosa is to analyze the mass spectrum characteristic peak of a strain by VITEK MS SARAMIS software by utilizing the identification model so as to distinguish the VIM and the SPM.
The invention has the beneficial effects that:
the invention analyzes the characteristic peak of the mass spectrum protein of VIM type and SPM type metalloenzyme pseudomonas aeruginosa by MALDI-TOF MS, and establishes a recognition model for distinguishing two subtypes; the identification model can quickly and accurately identify the VIM type and SPM type metalloenzyme pseudomonas aeruginosa, and has the advantages of simple and convenient operation, short time consumption and the like, so a brand new thought is provided for the research of identifying the bacterial subtype by MALDI-TOF MS, and the identification model has a very wide application prospect.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic representation of a common mass peak;
FIG. 2 is a schematic diagram of mass spectral peak classification;
FIG. 3 is a schematic view of a VIM model;
fig. 4 is a schematic diagram of a SPM type model.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example (b):
screening of Pseudomonas aeruginosa for metalloenzymes VIM-type and SPM-type
Firstly, non-repetitive pseudomonas aeruginosa 2687 separated in 2014-2017 of Dacron hospitals of army medical university, and screening imipenem drug-resistant pseudomonas aeruginosa 258 from the collected pseudomonas aeruginosa by an LIS system and WHONET 5.6 software. The selected strains are stored in an ultra-low temperature refrigerator at minus 80 ℃ by using strain tubes, and the types of specimens mainly comprise sputum, alveolar lavage, urine, pus, secretion, drainage fluid and the like. Then taking out the screened 258 pseudomonas aeruginosa strains from an ultralow temperature refrigerator at minus 80 ℃, taking out the bacteria from a freezing tube to be transferred to a Columbia blood agar plate after the pseudomonas aeruginosa strains are recovered to the room temperature, and placing the Columbia blood agar plate at 37 ℃ and containing 5 percent of CO2Incubating in an incubator for 24h, and rechecking the bacteria identification result by a VITEK-MS mass spectrometer. By the imipenem-EDTA paper method[1]And performing a primary screening test on the MBL phenotype to obtain 106 MBL phenotype positive strains. Then respectively carrying out PCR amplification on IMP, VIM, SPM, GIM, SIM and NDM genes by taking 106 MBL phenotype positive strain DNAs as templates, wherein each primer sequence is according to the literature[2-4]Design, see table 1. And (3) PCR system: taq PCR Master Mix system 12.5. mu.L, upstream and downstream primers 1. mu.L each, template DNA 1. mu.L, using ddH2Make up to 25. mu.L of O. And (3) PCR reaction conditions: pre-change at 94 DEG C5 min; denaturation at 94 ℃ for 30 s; annealing for 30s, wherein each annealing temperature is shown in a table 1; extension at 72 ℃ for 30s or 60s (depending on the product length); denaturation, annealing and extension for 30 cycles; extension was continued for 7min at 72 ℃. And (5) carrying out agarose gel electrophoresis on the amplification product at the mass concentration of 2.0%, and observing the result by using a gel imager. And finally, connecting the MBL gene positive PCR product to a TA carrier, sending the connecting carrier to Huada gene Limited company for sequencing, comparing the sequence in GenBank to determine 40 strains with positive VIM and 35 strains with positive SPM, wherein the sequencing analysis result is completely consistent with the electrophoresis result, and the positive IMP, SIM, GIM and NDM strains are not amplified.
TABLE 1 PCR primer sequences and product lengths
2. Extraction of protein from laboratory strains
Add 300. mu.L of ddH2O to the sterilized EP tube and select a single Pseudomonas aeruginosa colony (about 5-10 mg) specimen on the medium with ddH2Mixing O, standing for 1min, adding 900 μ L of anhydrous ethanol, shaking, mixing, standing for 1min, centrifuging at 13000rpm for 2min, and removing supernatant; adding 50 μ L of 70% formic acid into an EP tube, shaking and mixing uniformly, standing for 1min, adding 50 μ L of acetonitrile, shaking and mixing uniformly, standing for 1min, centrifuging at 13000rpm for 2min, sucking out supernatant protein extract for later use, and marking and numbering.
3. Collecting mass spectrum of experimental strain
Randomly selecting 15 VIM-type and 15 SPM-type library-establishing strains, and performing MALDI-TOF MS detection.
4. Clustering analysis of VIM-type and SPM-type metalloenzyme pseudomonas aeruginosa by using MALDI-TOF MS
And (3) introducing mass spectrum results of 15 VIM-type strains and 15 SPM-type strains into an SARAMIS database, and performing cluster analysis on mass spectrum peaks of the two strains to find out that partially different mass spectrum peaks exist between the two strains, which is shown in figure 1.
5. Establishing VIM type and SPM type metalloenzyme pseudomonas aeruginosa identification model database
The two mass spectral peaks were analyzed by comparison with LaunchPad software to obtain 17 high signal intensity peaks as mass peaks common to both (3601m/z,4434m/z,4544m/z,4849m/z,4992m/z,5114m/z,5210m/z,5737m/z,6046m/z,6348m/z,6676m/z,7203m/z,7595m/z,8786m/z,9089m/z,9987m/z and 10229m/z), see FIG. 2. In order to screen the characteristic peaks which are different between the VIM-type strain and the SPM-type strain, the LaunchPad software is used again to compare the mass spectrum characteristic peaks of the VIM-type strain and the non-VIM-type strain (including SPM-type and non-metallogenic pseudomonas aeruginosa) and the SPM-type strain and the non-SPM-type strain (including VIM-type and non-metallogenic pseudomonas aeruginosa), finally, 4 characteristic peaks (3230m/z,3409m/z,5450m/z,7329m/z) of the VIM-type strain and 3 characteristic peaks (3429m/z,5490m/z,7390m/z) of the non-VIM-type strain are selected as a model for identifying the VIM-type (figure 3), and 2 characteristic peaks (3663m/z,6264m/z) of the SPM-type strain and 2 characteristic peaks (4068m/z,4282m/z) of the non-SPM-type strain are selected as a model for identifying the SPM-type (figure 4).
6. Validating newly built identification model databases
25 strains of VIM-type and 30 strains of non-VIM-type (including 20 strains of SPM-type and 10 strains of non-metalloenzyme-producing) pseudomonas aeruginosa are selected to verify and establish a VIM-type recognition model, and 20 strains of SPM-type and 30 strains of non-SPM-type (including 25 strains of VIM-type and 5 strains of non-metalloenzyme-producing) pseudomonas aeruginosa are selected to verify and establish a SPM-type recognition model. Covering 1 mu L of supernatant protein extracting solution of each specimen on a target plate, uniformly smearing, covering 1 mu L of matrix solution after complete drying, uniformly smearing, putting the target plate into a VITEK MS mass spectrometer after complete drying for acquiring spectrum data, and comparing mass spectrum characteristic peaks of all verification strains with the two self-built identification models to obtain identification results of the newly-built identification models, namely sensitivity, specificity, positive predicted values and negative predicted values.
The result shows that the MALDI-TOF MS can correctly identify 23/25VIM type strains, the sensitivity is 92.0%, the specificity is 100.0%, the positive predictive value is 100.0%, and the negative predictive value is 93.7%. For SPM-type strains, the sensitivity was 80.0%, the specificity was 100.0%, the positive predictive values were 100.0%, and the negative predictive values were 88.2%, as detailed in table 2.
Table 2 results of two models for verifying and identifying VIM type and SPM type
Note: "- -" indicates the absence of this item.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
[ REFERENCE ] to
[1]Sachdeva R,Sharma B,Sharma R.Evaluation of different phenotypic tests for detection of metallo-β-lactamases in imipenem-resistant Pseudomonas aeruginosa.J Lab Physicians.2017.9(4):249-253.
[2]Tato M,Coque T M,Baquero F,et al.Dispersal of carbapenemase blaVIM-1gene associated with different Tn402 variants,mercury transposons,and conjugative plasmids in Enterobacteriaceae and Pseudomonas aeruginosa[J].Antimicrobial Agents and Chemotherapy,2010,54(1):320-327.
[3]Hentschke M,Goritzka V,Campos C B,et al.Emergence of carbapenemases in Gram-negative bacteria in Hamburg,Germany[J].Diagnostic Microbiology and Infectious Disease,2011,71(3):312-315.
[4]Vatcheva-Dobrevska R,Mulet X,Ivanov I,et al.Molecular epidemiology and multidrug resistance mechanisms of Pseudomonas aeruginosa isolates from Bulgarian hospitals[J].Microbial Drug Resistance,2013,19(5):355-361.
Claims (4)
1. A construction method of a VIM type and SPM type metalloenzyme pseudomonas aeruginosa identification model is characterized by comprising the following specific steps:
(1) firstly, introducing mass spectrum results of VIM-type and SPM-type strains into an SARAMIS database, and finding out partial different mass spectrum peaks between the VIM-type and SPM-type strains by clustering analysis of mass spectrum peaks of the VIM-type and SPM-type strains; then, the two mass spectrum peaks are compared and analyzed through LaunchPad software to obtain 17 high signal intensity peaks serving as the common mass peaks of the two;
(2) in order to screen characteristic peaks which are different between VIM type strains and SPM type strains, the launchPad software is used again to respectively compare the mass spectrum characteristic peaks of the VIM type strains and the non-VIM type strains as well as the mass spectrum characteristic peaks of the SPM type strains and the non-SPM type strains;
(3) finally, selecting 4 characteristic peaks of the VIM-type strain and 3 characteristic peaks of the non-VIM-type strain as models for identifying the VIM type, and selecting 2 characteristic peaks of the SPM-type strain and 2 characteristic peaks of the non-SPM-type strain as models for identifying the SPM type;
in step (1), the 17 high signal intensity peaks include:
3601m/z,4434m/z,4544m/z,4849m/z,4992m/z,5114m/z,5210m/z,5737m/z,
6046m/z,6348m/z,6676m/z,7203m/z,7595m/z,8786m/z,9089m/z,9987m/z and 10229 m/z;
in the step (3), 4 characteristic peaks of the VIM-type strain are as follows:
3230m/z,3409m/z,5450m/z,7329m/z, 3 characteristic peaks of non-VIM strains are 3429m/z,5490m/z,7390 m/z;
in the step (3), 2 characteristic peaks of the SPM type strain are 3663m/z and 6264m/z, and 2 characteristic peaks of the non-SPM type strain are 4068m/z and 4282 m/z.
2. The constructing method according to claim 1, wherein in the step (1), 15 strains of VIM type and 15 strains of SPM type are randomly selected and subjected to cluster analysis by sarasmis software on the number of peaks with the same mass of the two strains.
3. Use of the recognition model of claim 1 for the identification of pseudomonas aeruginosa by VIM-and SPM-type metalloenzymes.
4. A method for rapidly and accurately identifying VIM-type and SPM-type metalloenzyme pseudomonas aeruginosa, which is to analyze the mass spectrum characteristic peak of a strain by VITEK MS SARAMIS software by utilizing the identification model of claim 1 so as to distinguish the VIM-type and the SPM-type.
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