Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics

Abstract

Nucleic acid amplification techniques such as PCR have facilitated rapid and accurate diagnosis in central laboratories over the past years. PCR-based amplifications require high-precision instruments to perform thermal cycling reactions. Such equipment is bulky, expensive and complex to operate. Progressive advances in isothermal amplification chemistries, microfluidics and detectors miniaturisation are paving the way for the introduction and use of compact 'sample in-results out' diagnostic devices. However, this paradigm shift towards decentralised testing poses diverse technological, economic and organizational challenges both in industrialized and developing countries. This review describes the landscape of molecular isothermal diagnostic techniques for infectious diseases, their characteristics, current state of development, and available products, with a focus on new directions towards point-of-care applications.

Keywords: ASSURED; diagnostic; isothermal amplification; molecular test; point-of care.

Figure 1.

Loop-mediated isothermal amplification. For clarity, only the process initiated in one strand is shown. (A) Primer design with inner primers (F1c-F2 and B1c-B2) and outer primers (F3 and R3). Loop primers are not shown. (B) Amplification process. (1) F2 region of the FIP primer anneals with DNA template by its homologous sequence (F2c) and strand extension take place by the polymerase. (2) DNA polymerase synthesizes a new strand from F3 and displaces the previous DNA strand. (3) F1c segment of F3 primer hybridizes with F1. At other end, B3 and BIP primers hybridize with their homologues sequences and process 1 and 2 are repeated. (4) Formation of loop DNA molecule. (5) DNA molecule steam loops on both ends enter in a cycling step and a concatemere final product with several inverted repeats is produced (6).

Figure 2.

Helicase-dependent amplification. (A) Helicase unwinds the dsDNA. (B) SSB protein prevents premature strand reassociation. (C) Primer hybridizes with the target and is extended by the DNA polymerase while helicase continues unwinding the dsDNA remaining. (D) New dsDNA copy is formed as a product and is able to go into to a new cycle.

Figure 3.

Recombinase polymerase amplification. (A) Recombinase exchanges primers with the target DNA template. (B) SSB prevents premature strand reassociation while primes anneal with target DNA. (C) DNA polymerase synthesizes new DNA strands unwinding remaining dsDNA. (D) Two dsDNA molecules are generated and can again repeating the cycle.

Figure 4.

Smart amplification process v.2. MutS goes over template DNA strand, and DNA polymerase drives the extension (on top). If MutS find a mismatch (black arrow), it is blocked and both MutS nether DNA polymerase are able to continue the process (bottom).

Figure 5.

Isothermal and chimeric primer-initiated amplification of nucleic acid. For clarity, the process is shown only from one strand. (A and B) Polymerase extension from the chimeric primer produces a dsDNA molecule. (C) RNAseH nicks the original primer into the 3′ extreme. (D and E) Strand displacing polymerase extension from nick site release a shorter ssDNA that goes into the same process until generated molecule is too short.