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Published on: October 23, 2011
Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology
Andreas Nocker1, Alberto Mazza, Luke Masson
1Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA. andreas.nocker@gmail.com <andreas.nocker@gmail.com>
Journal of Microbiological Methods
|December 24, 2008
Summary
Propidium monoazide (PMA) treatment effectively distinguishes live from dead bacteria in diagnostic microarray analysis. This method inhibits DNA amplification from dead cells, improving the accuracy of bacterial detection in mixed samples.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Distinguishing live from dead bacteria is crucial for accurate molecular diagnostics.
- Current DNA-based methods struggle to differentiate viable from non-viable cells.
- Live cell detection is paramount for relevant diagnostic outcomes.
Purpose of the Study:
- To evaluate propidium monoazide (PMA) treatment for excluding dead bacterial cells from detection using diagnostic microarray technology.
- To assess the efficacy of PMA in combination with microarray analysis for bacterial mixtures.
- To improve the relevance of molecular diagnostic data by minimizing signals from membrane-compromised cells.
Main Methods:
- Utilized propidium monoazide (PMA) to selectively target membrane-compromised bacterial cells.
- Employed diagnostic microarray technology for detecting specific bacterial species (Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Serratia marcescens, Escherichia coli O157:H7).
- Amplified chaperonin 60 (cpn60) genes and hybridized PCR products to custom oligonucleotide probes on the microarray.
- Validated results using quantitative PCR (qPCR) to confirm PMA's inhibitory effect on DNA from killed cells.
Main Results:
- PMA treatment successfully inhibited the PCR amplification of DNA from isopropanol-killed bacterial cells in defined mixtures.
- Microarray analysis showed a significant reduction in detection signals when targeting killed cells, consistent with qPCR findings.
- The combination of PMA treatment and diagnostic microarray detection effectively minimized signals from dead cells.
Conclusions:
- Propidium monoazide (PMA) treatment is a suitable method for excluding dead cells in diagnostic microarray analyses of bacterial mixtures.
- This approach enhances the relevance of molecular diagnostic data, particularly in pathogen risk assessment.
- PMA treatment combined with microarray detection offers a valuable tool for accurate bacterial diagnostics.
