Isothermal nucleic acid amplification and its uses in modern diagnostic technologies

Abstract

Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.

Keywords: ASSURED; Isothermal DNA amplification; LAMP; Point of care device.

Fig. 1

Types of isothermal amplification techniques used for biosensing applications

Fig. 2

Schematic representation of the LAMP reaction. (I) Formation of dumbbell-shaped structure that starts from the FIP and BIP primers. (II) Cycling amplification of the dumbbell-shaped structure results in a stem–loop structure, which then (III) forms a cauliflower-like structure in the elongation and recycling step, resulting in different sizes of DNA amplicons producing a ladder pattern in agarose gel electrophoresis (reproduced with permission by Notomi et al. (2000), Copyright © 2000, Oxford University Press)

Fig. 3

(I) Schematic diagram of SARS-CoV 2 detection using crumpled graphene field effect transistor (cgFET) device. A Genome target site of SARS-CoV 2. B Workflow of sample collection and isothermal amplification for detection. C Crumpled graphene (cg) is used as a sensing material for FET and the chamber is used for isolating virus samples (reprinted with permission from Park et al. (2021), Copyright © 2021, American Chemical Society). (II) Droplet generation by inkjet printer. a–c Droplet generation from inkjet printer and its microscopic view (reprinted with permission from Fan et al. (2022), Copyright © 2022, MDPI, Basel, Switzerland). (III) Colorimetric detection of Mycobacterium tuberculosis from sputum sample using Au-NP. (1–3) DNA extraction and amplification through LAMP. (4) Preparation of Au-NP using HAuCl₄ and sodium citrate tribasic dihydrate. (5) Dia-ultrafiltration of the prepared AuNP, (6) MgSO₄ and MgCl₂ are added to induce aggregation in the absence of DNA. Detection through colorimetric method (reprinted with permission from Habiburrahman et al. (2021), Copyright © 2021, Journal of Infection in Developing Countries). (IV) Design of a turbidimeter for MgPO₄ quantification. (A) Turbidimeter consists of eight light emitting diodes and photodiodes and eight sample positions. (B) Orthographic view of the turbidimeter (reprinted with permission from Mori et al. (2004), Copyright © 2003 Elsevier B.V.)

Fig. 4

Schematic diagrams of LAMP reaction and working of NALFA strips. The flowchart shows: sample collection and thermal lysis for DNA extraction for LAMP. Amplified DNA was added to the NALFA strips to detect Salmonella and Staphylococcus aureus (reprinted with permission from Kim et al. (2022), Copyright © 2022, Elsevier)

Fig. 5

Methyltransferase (MTase)-assisted SDA technique for detection of MTase activity. Molecular beacons labeled with FAM and TAMRA as fluorophore for visual detection. Reprinted with permission from Cui et al. (2019). Copyright © 2019, Royal Society of Chemistry

Fig. 6

Working of fluorescent biosensor based on HDA for detection of β-glucosyltransferase. A β-GT catalyzes the transfer of β-glucosyl residue from UDP-glucose to form 5-ghmc. B Schematic diagram of fluorescent biosensor based on SYBR Green for detection of β-GT (reprinted with permission from Liu et al. (, Copyright © 2020, American Chemical Society)

Fig. 7

Detection of β-estradiol (E2) using electrochemical biosensor based on EXPAR and HCR ssDNA is produced through EXPAR. HCR signal output detects ssDNA using the turn off–turn on mechanism at 520 nm. Avidin-modified MNPs attached with biotin modified the E2-aptamer for detection of β-estradiol (E2) (reprinted with permission from Wang et al. (2020a, b). Copyright © 2020, Elsevier)

Fig. 8

Schematic diagram of SPIA. SPIA has a blocker, RNAse H and DNA polymerase. A, B DNA/RNA primer is first amplified at constant temperature where (C, D) RNAse H dissolves the RNA and extension continues. E, F A new DNA/RNA primer binds on the exposing part and extension continues. G Blocker stop the ssDNA amplification once the extension proceed to the blocker subsequently producing large number of ssDNA © 2020, Elsevier)