Helicase-dependent isothermal amplification: a novel tool in the development of molecular-based analytical systems for rapid pathogen detection

Abstract

Highly sensitive testing of nucleic acids is essential to improve the detection of pathogens, which pose a major threat for public health worldwide. Currently available molecular assays, mainly based on PCR, have a limited utility in point-of-need control or resource-limited settings. Consequently, there is a strong interest in developing cost-effective, robust, and portable platforms for early detection of these harmful microorganisms. Since its description in 2004, isothermal helicase-dependent amplification (HDA) has been successfully applied in the development of novel molecular-based technologies for rapid, sensitive, and selective detection of viruses and bacteria. In this review, we highlight relevant analytical systems using this simple nucleic acid amplification methodology that takes place at a constant temperature and that is readily compatible with microfluidic technologies. Different strategies for monitoring HDA amplification products are described. In addition, we present technological advances for integrating sample preparation, HDA amplification, and detection. Future perspectives and challenges toward point-of-need use not only for clinical diagnosis but also in food safety testing and environmental monitoring are also discussed. Graphical Abstract Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA).

Keywords: Helicase; Isothermal; Molecular test; Nucleic acid amplification; Pathogen detection.

Graphical Abstract

Expanding the analytical toolbox for the detection of DNA sequences specific of pathogens with isothermal helicase dependent amplification (HDA)

Fig. 1

Scheme of the helicase-dependent amplification system, adapted from reference [7]. (1) Unwinding dsDNA by helicase and stabilization of ssDNA by SSB; (2) annealing of primers, and (3) elongation of primers by DNA polymerase

Fig. 2

Right panel: schematic representation of HDA assays with homogeneous fluorescent monitoring, by using dual-labeled probes (a), TaqMan (b) hairpin ones. Left panel: a previous sequence-specific sample preparation employing RNA probes along with magnetic particles modified with an antibody specific for RNA-DNA hybrids boosts assay performance in clinical samples

Fig. 3

Schematic of lateral flow HDA assay (left), symmetric (right), asymmetric DNA amplification

Fig. 4

Hybridization-based on-surface capture of HDA amplicons: (1) homogeneous asymmetric HDA, (2) specific capture of biotinylated ss-amplicon, and (3) enzyme labeling and optical detection of the enzymatic product