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).

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…IA methods could become more popular than PCR soon, owing to their low energy requirement and simplicity which allows for their possible integration into simple, compact systems. 26 IA methods differ from one another in features such as the number of primers and enzymes, the temperature of amplification and template types used. Some POC platforms based on IA have been developed with some already commercialised (Table 2).…”

“…57,58 HDA is similar to the process of DNA replication in living organisms. 26,59 The process begins when the helicase enzyme disrupts the hydrogen bonds of dsDNA and separates them using energy derived from ATP hydrolysis. The resulting single-stranded DNA (ssDNA) is covered by single-strand binding proteins (SSBs) which stabilise the strand and prevents reannealing between the two separated strands ( Figure 2).…”

“…64 Amplified products from HDA can be visualised in real-time by fluorescence and by using agarose gel electrophoresis. 26,56 Some limitations of HDA include sample contamination, low reaction temperature, generation of non-specific amplification products due to primer mispairing and amplification inhibition. 26,65 However, continuous improvement of HDA can surmount these problems.…”

“…26,56 Some limitations of HDA include sample contamination, low reaction temperature, generation of non-specific amplification products due to primer mispairing and amplification inhibition. 26,65 However, continuous improvement of HDA can surmount these problems. Primers for HDA can be designed using PrimerQuestSM ® provided by Integrated DNA Technologies (Coralville, USA).…”

CelB is a suitable marker for rapid and specific identification of Klebsiella pneumoniae by the loop-mediated isothermal amplification (LAMP) assay

“…Although it has the advantages of easy operation and low cost, it was time-consuming and has low accuracy. With the rapid development of bioinformatics and molecular biology, conserved nucleic acid sequences with interspecies-specific and intraspecies commonality in the pathogens have been discovered one after another [8]. Based on this, an army of detection techniques has been established, such as the polymerase chain amplification (PCR), real-time fluorescent PCR, loop-mediated isothermal amplification (LAMP) [9][10][11].…”