| Summary: | 碩士 === 長榮大學 === 醫學研究所碩士班護理組 === 93 === ABSTRACT
The aim of this thesis was to establish the detection system for bacterial load on the disposable respiratory protective masks. The common bacteria caused in nosocomial infection were used as major index for analysis.
We first set up a quantitative system using the real time polymerase chain reaction (real-time PCR). The universal primers and probe were designed by Nadkarni et al. according to the unique and conservative 16S rDNA gene, which could identify more the 34 bacterial species. In our study, we showed the PCR system could detect Staphylococcus aureus, Escherichia coli, Pseudomonas amyloderamosa, Legionella pneumophila, Mycobacterium tuberculosis and also Candida pseudotropicalis. A standard curve set up by different concentration of purified Escherichia coli DNA were used to determine the bacterial number simultaneously. Along with the increasing PCR cycle, the fluorescence of reporter dye of the probe would increase. This system provided a rapidly and sensitively quantitative detection. Since masks may contain gram-positive and gram-negative bacteria, we established the DNA extraction system – water bath sonication. We showed that 40 min of indirect sonication is sufficient for lysis and release of DNA from gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli.
Using the mask containing a known number of Staphylococcus aureus or Escherichia coli, we can monitor and compare the elution effect. We showed 0.2% Tween 80 had better elution rate than 1% Triton X-100. However, the final result revealed that the number of Escherichia coli or Staphylococcus aureus on the mask eluting by Tween 80, releasing DNA by indirect sonication, and quantifying by real time PCR the 16S rDNA were 193% and 55% respectively，compared to the number calculated by plate count method. Although our preliminary study on the detection system for bacterial load on the disposable respiratory protective masks used by health care workers was not totally perfect, but it provides information for the future research in developing microorganism examination. Furthermore, integrated the data from the bacterial load examination of masks and the information collected by the questionnaire, it may also provide information for evaluating the expired time of wearing mask, infection control in the hospital, and the effectiveness and performance measures of environmental control equipment and approaches for the biologically hazardous areas.
Keywords: health care worker, disposable respiratory protection mask, real time PCR
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