Quantification of Staphylococcus aureus by propidium monoazide in combination with real-time quantitative PCR
Date Issued
2015
Date
2015
Author(s)
Lin, Meng-Hsuan
Abstract
Staphylococcus aureus, considered as one of the infectious bioaerosols, may be transmitted to the humans by airborne route. S. aureus plays an important role in nosocomial infections and has been detected in the residence, community and poultry feeding industry. Long-term uses of the antibiotics have caused the emergence of methicillin-resistant Staphylococcus aureus (MRSA) that was resulted from the drug resistance of S. aureus. Detecting and quantifying this pathogen via appropriate analytical method is necessary to identify the exposure risks of people, adopt effective intervention strategies and evaluate the performances of control measures. Monitoring a specified pathogen in environment may be accomplished via the analyses of samples with culture assay and a series of isolation/identification processes. However, such analytical methods are complex, time-consuming, limited by the number of workable colonies and can only detect culturable cells but not the cells with viable but not culturable (VBNC) state; therefore, the microbial quantification tends to be falsely determined. The real-time quantitative polymerase chain reaction (qPCR) detects the DNA of microorganism of a specific target and is not affected by the nonculturability of target cells. Thus, this qPCR method overcomes the drawback of the culture assay. However, the qPCR may overestimate the number of viable cells because it cannot differentiate the dead cells from viable ones. To address the issue that both culture assay and qPCR may not accurately quantify the exposure level of S. aureus, this study is initiated to develop a qPCR-based method coupled with nucleic acid dye that can accurately quantify viable S. aureus. Different types and concentrations of nucleic acid dye (propidium monoazide) are evaluated and the detection limit of this assay are determined. Furthermore, this analytical method will be validated in environments. It is expected that this study will help to detect and quantify the exposure of S. aureus in environments precisely. According to the experiments by culture assays and BacLight/epifluorescence microscopy, our data show heating cells at 90℃ for 20 min resulted in a complete loss of cell culturability and significant damages on the integrity of cell membrane. Further examination of PMA-treated cells under light and epifluorescence microscopes indicate that PMA penetrated cells pre-heated at 90℃ for 20 min (represented as dead S. aureus). However, it is noted that PMA (23 μg/mL) did not penetrate the membrane of some alive cells. The performance of five different concentrations of PMA (1.5, 2.3,10, 23, 46 μg/mL) coupled with the qPCR was further assessed. For heat-inactivated S. aureus, PMA significantly inhibited the DNA amplification in qPCR. As for live S. aureus, only the PMA of 1.5 and 2.3 μg/mL showed comparable DNA concentrations between PMA-treated and –untreated cells (P>0.05). The PMA of 1.5 and 2.3 μg/mL were tested for the linearity, and obtained the respective R2 values of 0.97 and 0.96 for live cells in the range of 103-108 cells/mL.
Subjects
Staphylococcus aureus
real-time quantitative PCR
Type
thesis
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