Isothermal amplification methods amplify nucleic acids at a constant temperature, eliminating the need for a thermal cycler. They are increasingly used in point-of-care diagnostics, field testing, and resource-limited settings.

LAMP Overview

Loop-mediated isothermal amplification (LAMP) uses 4–6 primers that recognize 6–8 distinct regions on the target sequence. The reaction is performed at 60–65 °C using a DNA polymerase with strand-displacement activity (Bst polymerase from Bacillus stearothermophilus). The polymerase continuously displaces newly synthesized strands, creating loop structures that serve as templates for further amplification.

The result is exponential amplification that produces up to 10⁹ copies in under an hour. Because of the complex primer set and the branched amplification products, LAMP is extremely specific — it can discriminate single-nucleotide differences.

LAMP Primer Design

A LAMP primer set includes:

• FIP (Forward Inner Primer): contains F2 and F1c sequences
• BIP (Backward Inner Primer): contains B2 and B1c sequences
• F3 and B3 (outer primers): initiate strand displacement
• Optional Loop primers (LF and LB): accelerate the reaction by priming loop structures

Primer design is critical and is typically done with dedicated software (PrimerExplorer, LAMP Designer). The target region should be 200–300 bp.

Detection Methods

LAMP products can be detected by:

• Turbidity: the large amount of magnesium pyrophosphate precipitate formed during amplification is visible to the naked eye.
• Colorimetric dyes: phenol red or hydroxynaphthol blue change color when the reaction pH changes or magnesium is consumed.
• Fluorescence: intercalating dyes (SYBR Green, EvaGreen) or calcein produce a fluorescent signal.
• Lateral flow: using labeled primers that incorporate haptens for immunochromatographic strip detection.

Other Isothermal Methods

• RPA (Recombinase Polymerase Amplification): uses a recombinase to pair primers with homologous duplex DNA, a single-stranded DNA binding protein to stabilize the displaced strand, and a strand-displacing polymerase. It operates at 37–42 °C and produces detectable product in 5–20 minutes. RPA is the fastest isothermal method and is compatible with freeze-dried formats.
• HDA (Helicase-Dependent Amplification): uses a DNA helicase to unwind the double helix instead of heat denaturation, operating at 37–65 °C depending on the helicase.
• NASBA (Nucleic Acid Sequence-Based Amplification): amplifies RNA targets at 41 °C using three enzymes (reverse transcriptase, RNase H, and T7 RNA polymerase), producing RNA amplicons.

Applications

Isothermal amplification is widely used for pathogen detection (SARS-CoV-2, malaria, tuberculosis, Zika virus), food safety testing, and environmental monitoring. The ability to run reactions in a water bath or heat block and detect results by color change makes it ideal for decentralized testing.