| Summary: | 碩士 === 慈濟大學 === 醫學生物技術研究所 === 98 === Abstract
The rapid and precise identification of bacterial pathogens is significant for
the patient supervision and initiation of appropriate antibiotic therapy in the early
stages of infection. Recently, CE and mass spectrometry has been used widely as a
diagnostic tool in separation and identification of microbial mixtures. Furthermore,
most of the human normal flora is composed of anaerobic bacteria and can infect
deep wounds, deep tissues, internal organs and also inflict occasionally
life-threatening disease. In the present investigation, we performed two analytical
approaches for rapid identification of pathogens. At first, we present an off-line
coupling of capillary electrophoresis (CE) to matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
approach for fast identification of anaerobes. In this study, we successfully
separated and collected several anaerobic pathogens like Fusobacterium
nucleatum, Bacteroides fragilis, Prevotella bivia, Clostridium sporgenes,
Clostridium difficile and Porphyromonas gingivalis from clinical specimens
performing CE and then analyzed by MALDI-TOF mass spectrometry. Different
pathogens were further identified shortly through protein fingerprinting approach.
On the other hand, we performed HPLC-Nanospray-MS/MS analysis for
identification of anaerobic pathogens. This technique is most useful for microbial
identification because of its sensitivity, high throughput capacity and it is readily
amenable to automation. Here, we developed a new method to identify bacteria
based on data-dependent tandem mass spectrometry. We used Bioworks browser
with SEQUEST search engine combined NCBI BLAST proteome database,
followed ranking peptide by its score and found specific unique peptide of the
bacterium to identify unknown pathogen. In conclusion, both the approaches allow
the rapid and efficient separation and analyzed the pathogens within one day.
Moreover it was easy, faster and almost automated and offer clear advantages over
conventional methods. Furthermore, the offline coupled CE-MALDI-TOF-MS
approach allows reinvestigation of the collected fractions by other methods
following the mass spectrometric analysis.
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