LAMP Test: Rapid Detection Method for Infectious Agents

Detecting infectious agents quickly and accurately is crucial for controlling disease outbreaks and guiding treatment. Traditional diagnostic methods, such as PCR, can be time-consuming and require specialized equipment, making them less practical in resource-limited settings.

To address these challenges, researchers developed the loop-mediated isothermal amplification (LAMP) test, a rapid and sensitive method for detecting pathogens.

Reaction Mechanism

The LAMP test enables efficient DNA amplification under constant temperature conditions, typically between 60–65°C. Unlike PCR, which requires thermal cycling, LAMP relies on the strand-displacing activity of Bst DNA polymerase, eliminating the need for repeated heating and cooling cycles. This enzyme continuously synthesizes new DNA strands while displacing existing ones, allowing rapid amplification.

A key feature of LAMP is its use of six primers targeting distinct regions of the target sequence. The inner primers (FIP and BIP) initiate strand displacement and form loop structures that drive continuous amplification. The outer primers (F3 and B3) contribute to initial strand displacement, while loop primers (LF and LB) can further accelerate the reaction. This intricate primer design enhances specificity and speed.

As the reaction progresses, displaced single-stranded DNA forms loop structures that serve as new templates, leading to rapid DNA accumulation, often within 30 minutes. The resulting DNA structures, including stem-loop configurations and cauliflower-like concatemers, differ from the linear amplicons produced by PCR. These unique structures contribute to LAMP’s high sensitivity, minimizing nonspecific byproducts.

Reaction Components

The efficiency of LAMP depends on a carefully designed reaction mixture. At its core is Bst DNA polymerase, a thermostable enzyme from Bacillus stearothermophilus with strong strand displacement activity. Unlike Taq polymerase, Bst functions efficiently at a single temperature, enabling robust amplification in a shorter timeframe. Variants like Bst 2.0 and Bst 3.0 enhance reaction speed and tolerance to inhibitors, making them useful for point-of-care diagnostics.

Primer design is critical, with six primers targeting multiple regions of the DNA sequence. The inner primers (FIP and BIP) initiate amplification by introducing complementary sequences that facilitate loop formation. The outer primers (F3 and B3) assist in the initial strand displacement, while loop primers (LF and LB) further accelerate amplification. This design ensures high specificity and minimizes false positives.

The buffer system maintains optimal reaction conditions. Magnesium sulfate (MgSO₄), typically at 4–8 mM, stabilizes the polymerase and facilitates nucleotide incorporation. Tris-HCl maintains pH around 8.8, while potassium chloride (KCl) or ammonium sulfate (NH₄)₂SO₄ modulates ionic strength, influencing primer binding and reaction efficiency. The buffer composition is optimized for each assay to maximize speed while preventing nonspecific interactions.

Deoxynucleotide triphosphates (dNTPs) serve as DNA building blocks. These nucleotides—dATP, dTTP, dCTP, and dGTP—must be supplied at 0.8–1.4 mM each to sustain continuous polymerization. Maintaining an optimal dNTP balance is essential for achieving rapid, reliable results.

Steps in the Procedure

A LAMP assay begins with preparing the reaction mixture, ensuring optimal concentrations of all components. The DNA or RNA template, extracted from a clinical or environmental sample, must be sufficiently pure to avoid polymerase inhibition. Some point-of-care applications incorporate crude sample processing to bypass extensive purification.

Once the template is ready, it is combined with the reaction mix, including Bst DNA polymerase, primers, dNTPs, and buffer components. The primer set, designed to recognize multiple target regions, is added at carefully optimized concentrations to maintain specificity while maximizing amplification speed.

The reaction mixture is then incubated at a constant temperature, typically 60–65°C, using a water bath, heating block, or isothermal amplification device. Unlike PCR, which requires thermal cycling, LAMP proceeds continuously due to Bst polymerase’s strand displacement activity. DNA synthesis begins rapidly, generating a cascade of amplification products, often reaching detectable levels within 15–30 minutes. To prevent contamination, sealed reaction tubes and dedicated workspaces are used, as LAMP’s high DNA yield increases carryover risk.

Once amplification is complete, the reaction is terminated by cooling the tubes or adding reagents that inhibit polymerase activity. This prevents further DNA synthesis and stabilizes reaction products for analysis. Detection methods vary, with some assays using real-time fluorescence monitoring, while others rely on visual color changes or gel electrophoresis. The final analysis determines pathogen presence, providing rapid diagnostic results without extensive post-processing.

Methods for Detecting Amplification

Detecting successful amplification in a LAMP assay relies on clear, rapid readouts. One common technique is colorimetric detection using pH-sensitive dyes like phenol red or hydroxynaphthol blue (HNB). As DNA synthesis progresses, dNTP consumption and pyrophosphate release alter the reaction’s pH, triggering a visible color change. This approach is particularly useful for point-of-care diagnostics, as it requires no specialized equipment and allows results to be interpreted visually.

Fluorescence-based detection involves intercalating dyes like SYBR Green I or calcein, which bind to double-stranded DNA and emit fluorescence upon excitation. This enables real-time monitoring of amplification. Quantitative LAMP (qLAMP) has been used in clinical diagnostics for viral detection, including SARS-CoV-2, where fluorescence intensity correlates with viral load. This method enhances sensitivity while reducing false positives, as fluorescence is emitted only in the presence of amplified target sequences.

Turbidity measurement offers another detection method, utilizing the precipitation of magnesium pyrophosphate as a byproduct of DNA synthesis. As the reaction progresses, the accumulation of this insoluble compound increases solution turbidity, which can be measured using a spectrophotometer or observed visually. This method has been validated for bacterial infections such as Mycobacterium tuberculosis, demonstrating its utility in resource-limited settings. While less precise than fluorescence-based approaches, turbidity detection remains practical for large-scale screenings due to its simplicity and cost-effectiveness.