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Objective To evaluate the value of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) in detecting carbapenemases-producing Enterobacteriaceae. Methods Sixty-one carbapenemase-producing Enterobacteriaceae (CPE) and 108 carbapenemase-none-producing Enterobacteriaceae(NCPE) strains that isolated in the Renmin Hospital of Wuhan University from February 2016 to February 2017 were used in this study. Twenty CPE strains and 20 NCPE strains were se-lected and used to establish the optimum reaction system of MALDI-TOF MS for the detection of carbapene-mase-producing Enterobacteriaceae strains. Results The optimal reaction system of MALDI-TOF MS was successfully established:equal volumes of imipenem solution (1 mg/ml) and bacterial suspension (2 Mc-Farland turbidity standard) were first mixed together and incubated at 37℃ for 20 min,and then the super-natant was detected by MALDI-TOF MS after centrifugating. It would be a carbapenemase-producing strain if the peak of (300±0.55) m/z in the mass spectrum of imipenem disappeared completely. MALDI-TOF MS was used to identify carbapenemase-producing Enterobacteriaceae strains among the 169 strains and the result was consistant with that by using carbapenemase gene detection. Conclusion MALDI-TOF MS could accu-rately and rapidly detect carbapenemases-producing Enterobacteriaceae. It could be used as a routine method to provide references for early anti-infective treatment and infection control in hospitals.
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Objective To study the serum peptide fingerprint using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) technology,and find the different peaks with potential significance and establish the diagnosis model of Staphylococcus aureus and Escherichia coli bloodstream infection.Methods To establish ICR mice model of S.aureus and E.coli bloodstream infections,and collect serum samples.The serum samples were purified by weak cation exchange beads,the serum peptide fingerprint was recognized by using MALDI-TOF MS and BioExplorer software between infections group and normal control group.Results Compared with the normal control group,6 peptides were up-regulated,7 peptides downregulated and 8 peptides up-regulated first and then down-regulated in S.aureus infection group;And 5 peptides down-regulated,4 peptides down-regulated first and then up-regulated,and 8 peptides up-regulated first and then down-regulated in E.coli infection group.Conclusion MALDI-TOF MS combined with BioExplorer software may be used as a tool to study the serum peptides of S.aureus and E.coli bloodstream infection,effectively find significant peptides for establishing a diagnosis model of these two bacterial infections,and has a certain value for the diagnosis of bacterial bloodstream infection.
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In order to optimize the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) method for Salmonella identification,study the target board pollution situation of different pretreatment methods,and establish a safe and effective method for detection of Salmonella,we applied MALDI-TOF-MS technique to detect 4 standard strains-of Salmonella under different culture conditions and sample preparation method,and established optimization program.MALDI target board was detected under different preparation treatments.The optimization method was used to detect 33 strains Salmonella which isolated from diarrhea patients' stool.Identification results were compared with serological results.The study found that MALDI-TOF-MS method could accurate identify of Salmonella in different pretreatment methods and culture medium.Thirty-three strains of Salmonella identified by MALDI-TOF-MS method were all accurate appraisal in genus level,19 strains of them were completely consistent with the serotyping identification results,14 strains of them were not consistent with the serotyping identification results.There was no bacteria growth on the target board in different pretreatment methods.MALDI-TOF-MS method can in a fast,convenient,safe and accurate way identify Salmonella,and it can become an effective means for identification of Salmonella.
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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)is a powerful tool for routine identification of a variety of microorganisms,including bacteria,yeast and filamentous fungi.Fast,accurate, high-throughput and low-cost characteristics,and gradually used for filamentous fungal infection in the laboratory diagnosis. In the MALDI-TOF MS analysis of filamentous fungi,from sample preparation to obtaining accurate identification results, each step determines whether the medical technician can provide accurate results for the clinician.This review will provide a systematic overview of the applications and prospects of filamentous fungal analysis (including identification,typing,and an-tifungal susceptibility testing)from preparation of the test sample to the MALDI-TOF MS technique,as well as a review of some of its future developments.
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BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and accurate identification of clinical yeast isolates. In-tube formic acid/acetonitrile (FA/ACN) extraction is recommended prior to the analysis with MALDI Biotyper, but the direct on-plate FA extraction is simpler. We compared the Biotyper with the VITEK MS for the identification of various clinically relevant yeast species, focusing on the use of the FA extraction method. METHODS: We analyzed 309 clinical isolates of 42 yeast species (four common Candida species, Cryptococcus neoformans, and 37 uncommon yeast species) using the Biotyper and VITEK MS systems. FA extraction was used initially for all isolates. If ‘no identification' result was obtained following the initial FA extraction, these samples were then retested by using FA (both systems, additive FA) or FA/ACN (Biotyper only, additive FA/ACN) extraction. These results were compared with those obtained by sequence-based identification. RESULTS: Both systems correctly identified all 158 isolates of the four common Candida species after the initial FA extraction. The Biotyper correctly identified 8.7%, 30.4%, and 100% of 23 C. neoformans isolates after performing initial FA, additive FA, and FA/ACN extractions, respectively, while VITEK MS identified all C. neoformans isolates after the initial FA extraction. Both systems had comparable identification rates of 37 uncommon yeast species (128 isolates), following the initial FA (Biotyper, 74.2%; VITEK MS, 73.4%) or additive FA (Biotyper, 82.0%; VITEK MS, 73.4%). CONCLUSIONS: The identification rate of most common and uncommon yeast isolates is comparable between simple FA extraction/Biotyper method and VITEK MS methods, but FA/ACN extraction is necessary for C. neoformans identification by Biotyper.
Subject(s)
Candida , Cryptococcus neoformans , Mass Spectrometry , Methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , YeastsABSTRACT
Objectire To identify specific biomarkers that could improve early diagnsis of lung adenuearcinoma using matrix-assisted laser desorptian/ionization (MALDI) technology. Methods Serum samples were isolated from 17 patients with stage I lung adenuearcinoma and 17 age- and sex-matched healthy controls, and the serum proteomic profiles were obtained by matrix-assistcd laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results Compared with healthy control group, two highly expressed potential biomarkers were identified with the relative molecular weights of 6 631.64 Da and 4 964. 21 Da. The two best novel protein peaks were automatically chosen for the system training and the development of the constructed model. The constructed model was then used to test an independent set of masked serum samples from 15 lung adenocarcinoma patients and 22 healthy individuals. The analysis yielded a sensitivity of 93.3 %, and a specificity of 95.5 %. Conclusion These results suggest that MALDI-TOF-MS ProteinChip technology is a quick, convenient, and high-output analyzing method that is capable of selecting several relatively potential biomarkers from the serum of lung adenocarcinoma patients and may have a clinical value in the future, and will provide clues to identifying new serologic btomarkers of lung adenocarcinoma.
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Objective: To identify specific biomarkers that could improve early diagnosis of lung adenocarcinoma using matrix-assisted laser desorption/ionization (MALDI) technology. Methods: Serum samples were isolated from 17 patients with stage I lung adenocarcinoma and 17 age- and sex-matched healthy controls, and the serum proteomic profiles were obtained by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results: Compared with healthy control group, two highly expressed potential biomarkers were identified with the relative molecular weights of 6631.64 Da and 4964.21 Da. The two best novel protein peaks were automatically chosen for the system training and the development of the constructed model. The constructed model was then used to test an independent set of masked serum samples from 15 lung adenocarcinoma patients and 22 healthy individuals. The analysis yielded a sensitivity of 93.3%, and a specificity of 95.5%. Conclusion: These results suggest that MALDI-TOF-MS ProteinChip technology is a quick, convenient, and high-output analyzing method that is capable of selecting several relatively potential biomarkers from the serum of lung adenocarcinoma patients and may have a clinical value in the future, and will provide clues to identifying new serologic biomarkers of lung adenocarcinoma.
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Objective:To establish the method for the expression,purification and characterization of metallo-?-lactamases(IMP-1) and to explore the influence of experimental conditions on its separation and purification.Methods:pET9a/d-IMP-1 plasmid was transformed into E.coli competent BL21(DE3) cell and inoculated in LB medium.The supernatant were applied to SP Sepharose Fast Flow column,following by Sephadex G-75 column,and the purified enzymes were identified by SDS-PAGE and MALDI-TOF-MS and their activity was determined using Nitrocefin as substrate.Results:The purified enzyme showed high catalytic activity in the hydrolysis of most antibiotics.Its Michaelis constant K_m9.28?mol/L,and molecular weight Mr equalled 25111.9(determined by MALDI-TOF-MS).Conclusion:The method was established for the expression,purification and characterization of metallo-?-lactamases(IMP-1),which can be applicable to other metallo-?-lactamases.