Challenges Using Droplet Digital PCR for Environmental Samples

Droplet digital polymerase chain reaction (ddPCR) is a method used to detect and quantify nucleic acids even when present in exceptionally low numbers. While it has proven be valuable for clinical studies, failed widely adopted environmental studies but despite some limitations, ddPCR may represent better option than classical qPCR samples. Due the complexity of chemical biological composition samples, protocols tailored are not appropriate, results difficult interpret. We DNA samples originating from field determine protocol Samples included soils which had been inoculated with soil fungus Rhizophagus irregularis (environmental positive control), that targeted was naturally negative root both categories. To control effect inhibitors, we also an organismal extracted pure fungal spores (organismal control). Finally, no-template consisting only PCR reagents nuclease free water instead template DNA. Using original data, examined factors contribute poor resolution propose best practices ensure accuracy repeatability. Furthermore, evaluated manual automatic threshold determination methods novel based on multiple controls more appropriate