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Published on: February 23, 2018
Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell
Jessica K van Frankenhuyzen1, Jack T Trevors, Hung Lee
1School of Environmental Sciences, University of Guelph, Guelph, ON, Canada. jvanfran@uoguelph.ca
Journal of Microbiological Methods
|October 4, 2011
Summary
Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) offers a promising method for accurately detecting viable pathogens in biosolids. This technique overcomes limitations of traditional methods by differentiating between live and dead cells for improved microbial enumeration.
Area of Science:
- Environmental microbiology
- Molecular biology
- Wastewater treatment
Background:
- Sewage sludge, after treatment, becomes biosolids, which can be applied to land if regulatory standards are met.
- Traditional live-culture methods for pathogen detection in biosolids may underestimate viable microorganisms, particularly viable but non-culturable (VBNC) cells.
- Real-time quantitative polymerase chain reaction (qPCR) is a sensitive molecular tool but cannot distinguish between DNA from viable and non-viable cells.
Purpose of the Study:
- To review the application of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) for enumerating viable microorganisms in biosolids.
- To discuss the advantages of PMA-qPCR over traditional methods for pathogen detection in biosolids.
- To provide recommendations for optimizing the PMA-qPCR method for biosolids analysis.
Main Methods:
- Coupling qPCR with propidium monoazide (PMA), a DNA-binding dye that inhibits amplification of DNA from dead or membrane-compromised cells.
- Reviewing existing literature on the use of PMA-qPCR for microbial enumeration in various matrices, including biosolids.
- Identifying key parameters for method optimization, such as DNA extraction, sample turbidity, reagent concentration, and light exposure.
Main Results:
- PMA-qPCR effectively distinguishes between viable and non-viable cells by preventing the amplification of DNA from dead cells.
- The combination of PMA and qPCR offers a more accurate assessment of viable pathogen loads in biosolids compared to traditional culture-based methods.
- Successful application of PMA in other matrices suggests its potential efficacy in biosolids analysis.
Conclusions:
- PMA-qPCR is a suitable and advanced approach for the accurate enumeration of viable microorganisms in biosolids.
- Optimization of DNA extraction, sample preparation, and reaction conditions is crucial for reliable PMA-qPCR results.
- This method enhances regulatory compliance and public health safety concerning land application of biosolids.
