🔬 Loop-Mediated Isothermal Amplification (LAMP)

🧬 A Rapid and Accessible DNA Amplification Tool for MAP Detection

The detection of pathogens, such as Mycobacterium avium subsp. paratuberculosis (MAP), the bacterium that causes paratuberculosis in ruminants, has been transformed by the potent molecular technique known as Loop-Mediated Isothermal Amplification, or LAMP.

In contrast to traditional PCR, which uses repeated cycles of heating and cooling, LAMP amplifies DNA at a fixed temperature, enabling quicker and more flexible pathogen detection with less equipment.

LAMP is particularly useful in veterinary and agricultural settings where quick on-site diagnostics can reduce the spread of bacteria and enhance herd management.

🧫 The principle of LAMP

Primers are designed to target six different regions of the gene of interest in the LAMP (Loop-mediated Isothermal Amplification) method. Internal primers (FIP and BIP) and external primers (F3 and B3) are the two types of primers used in this technique. The external primers facilitate strand displacement during the reaction, whereas the internal primers are in charge of creating stem-loop structures.  

In particular, the complementary regions F2c and F3c on the template strand are where the FIP primer—which is made up of the FIc and F2 sequences—and the F3 primer, respectively, hybridize. These primers are extended when DNA polymerase is added, which creates a stem-loop structure. The complementary F1c and F1 sequences at the 5′ end of the FIP-linked strand enable this structure.

Then, the BIP (B1c + B2) and B3 primers use this freshly created FIP-linked strand as a template. A DNA molecule with a dumbbell shape and stem-loop structures at both ends is produced as a result of the reaction. The starting template for the next LAMP amplification cycle is this dumbbell-shaped DNA. It drives the production of double-length stem-loop DNA products by using itself as a template. The reaction creates DNA strands with multiple repeats of the target sequence inside stem-loop structures through continuous synthesis and strand displacement.

🔍 How LAMP Differs from PCR:

🎯 LAMP for MAP Identification

LAMP has shown promise in MAP diagnostics for identifying MAP DNA in tissue, milk, feces, and soil samples. Because the IS900 gene is specific for MAP, it is frequently used as a target. Additionally investigated are other gene markers such as f57, hspX, and dnaA.

Due to MAP's slow growth and difficulty in culture, LAMP provides a quick substitute for MAP in animal infection detection, enhancing early detection and farm biosecurity.

🧰 Sample Simplicity & Preparation

Sample preparation is minimal for LAMP. Simple procedures can be used for DNA extraction, and simple heating apparatus (such as a portable heater or water bath) can be used for the assay. The following methods allow for detection:

• Colorimetric dyes, such as Hydroxy Naphthol Blue
• Fluorescent indicators
• Dipsticks with lateral flow

✅ Advantages:

• Quick results in under an hour
• No need for thermocyclers
• High specificity and sensitivity
• Suitable for field deployment