Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

Abstract

Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

Rapid diagnosis of Zika virus through saliva and urine by Loop-mediated isothermal amplification (LAMP)

“…The fact that LAMP does not require thermal cycling makes the assay simple, fast and cost-effective compared with PCR assay [21,22]. …”

Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1

“…Although specific and rapid, those assays require well equipped laboratory and trained personnel. Recently, various isothermal amplification assays for diagnosis of infections with human and animal pathogens were designed (Zanoli et al 2012), allowing for the rapid and specific diagnostic with little need of equipment. The aim of our study was to design and evaluate two variants of loop-mediated isothermal amplification (LAMP) assays specific for gD and gE of BoHV-1 as a simple diagnostic tool.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…These include recombinase polymerase amplification, rolling circle amplification and helicase-dependent amplification (Zanoli and Spoto 2013). Finally, Notomi et al (2000) successfully devised a novel isothermal amplification method to amplify a limited amount of DNA copies into a million copies within an hour.…”