Critical review of methods for isothermal amplification of nucleic acids for environmental analysis

Abstract

The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance of a single temperature throughout the amplification reaction, which could facilitate deployment of IAMs in a variety of environmental and field settings. This review summarizes the mechanisms of the most common IAM methods and their use in studies of pathogens, harmful algae and fecal indicators in environmental waters, feces, wastewater, reclaimed water, and tissues of aquatic animals. Performance metrics of sensitivity, specificity and limit of detection are highlighted, and the potential for use in monitoring and regulatory contexts is discussed.

Keywords: Fecal indicators; Harmful algal blooms; LAMP; NASBA; Pathogens; Water quality; qPCR.