Abstract
Molecular diagnostic tools in the field of food and water quality analysis are becoming increasingly widespread. Usually, based on DNA amplification techniques such as polymerase chain reaction (PCR), these methods are highly sensitive and versatile but require well-equipped laboratories and trained personnel. To reduce analysis time and avoid expensive equipment, isothermal DNA amplification methods for detecting various target organisms have been developed. However, to make molecular diagnostics suitable for low-resource settings and in-field applications, it is crucial to continuously adapt the working steps associated with DNA amplification, namely sample preparation, DNA extraction, and visualization of the results. Many novel approaches have been evaluated in recent years to tackle these challenges, e.g., the use of ionic liquids for the rapid isolation of nucleic acids from organisms relevant for food and water analysis or the integration of entire analytical workflows on microfluidic chips. In any event, the future of applications in the field of isothermal amplification will probably lie in ready-to-use cartridges combined with affordable handheld devices for on-site analysis. This trend article aims to make prospective users more familiar with this technology and its potential for moving molecular diagnostics from the laboratory to the field.Graphical abstract
Highlights
The state-of-the-art and recent advances of isothermal DNA amplification methods for food and water analysis are presented by providing a broad overview on promising new developments and existing challenges
We focus on the latest scientific research and give an insight into what has been made commercially available far. These aspects are illustrated by a case study on the implementation of an isothermal amplification method for microbiological water quality analysis
7 TU Wien, Institute of Chemical, Environmental & Bioscience Engineering, Research Area Biochemical Technology, Research Group of Environmental Microbiology and Molecular Diagnostics, 1060 Vienna, Austria improvements are proposed that are needed to realize the vision of cost-effective molecular diagnostic tools for field applications
Summary
The state-of-the-art and recent advances of isothermal DNA amplification methods for food and water analysis are presented by providing a broad overview on promising new developments and existing challenges. To increase the experimental transparency of such molecular diagnostic tests, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were defined in 2009 [1] These guidelines aim to ensure the reliability of the results by describing the minimum information required to evaluate newly developed qPCR assays, thereby promoting consistency among different laboratories. The methods should be readily applicable by non-molecular biology trained personnel, indicating that the number of pipetting steps is minimized, and handling is kept intuitive to avoid sources of error These requirements were, for the first time, officially summarized by the World Health Organization (WHO) in their ASSURED guidelines for point-of-care testing (Affordable, Sensitive, Specific, User-friendly, Robust and rapid, Equipment-free, Deliverable to those who need them) [6]. They are based on different reaction principles, all methods have in common that the amplification of the targeted gene occurs
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