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Challenges And Applications In Using qPCR and Digital PCR For Environmental Samples

qPCR, Digital PCR, Microbial Source Tracking

The detection of the origin of faecal pollution in complex watersheds is beginning to take a prominent place in hazard identification and risk management policies. 

Microbial source tracking (MST) is a field that has developed molecular methods to differentiate various sources of faecal contamination from humans and animals. Tiong Gim Aw’s investigation involves next generation sequencing technology and metagenomics approaches on linking water security, microbial ecology and advanced biotechnology. His work examines new molecular methods for waterborne pathogens and quantitative microbial source tracking techniques.

What’s your research about?

Most of the MST methods utilise genetic markers, which are host-specific or library-independent, and mainly rely on quantitative PCR without cultivation of the microorganisms.

In my work, I use qPCR and most recently digital PCR to monitor microbial water quality including detection of pathogens in water for microbial source tracking to identify sources of faecal pollution in water.

What are the main challenges you face working with qPCR and dPCR?

There are currently three major challenges:

  • The need for a simple and efficient sample preparation method including concentration method to sample low abundance of pathogens such as viruses in water for qPCR and digital PCR.
  • The presence of PCR inhibitors such as humid acid in environmental water samples can increase an assay’s limit detection and cause false-negative results.
  • qPCR and digital PCR measure only DNA/RNA. Hence, they cannot be used to determine viable versus dead cells which is critical for public health risk. There is a need to develop a method coupled with qPCR and digital PCR for measuring only viable cells such as using ethidium monoazide (EMA) and propidium monoazide (PMA).
What has been the impact of these PCR methods to your work?

The main impact has been in both the clinical and environmental diagnostics sectors, particularly in pathogen detection and identification.

In terms of development, one of the most important one PCR techniques brought to my field is the ability to detect and measure pathogens that still cannot be cultured using traditional culture methods, for example noroviruses.

Furthermore, the development of digital PCR which allows absolute quantification of target genes without the use of standard DNA and has provided more sensitive and precise quantification of pathogens in both clinical and environmental samples.

Tiong, qPCR, PCR, digital PCR, watersheds, microbial source tracking

Tiong Gim Aw
is a Research Associate in the Department of Fisheries and Wildlife at Michigan State University. He participated at the 2nd qPCR and Digital PCR Congress: USA.


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