LAMP Assay Not Working? Master Primer Design First!

2026-03-13

Loop-Mediated Isothermal Amplification (LAMP) is a gene amplification technology invented by Japanese scholar Notomi in 2000. It is simple, rapid and accurate, enabling fast amplification of target DNA/RNA under isothermal conditions at 60–65 °C.

This technique requires 4–6 specific primers targeting 6–8 regions of the target gene, including outer primers (F3, B3), inner primers (FIP, BIP), and optional loop primers (Loop F, Loop B). With the strand-displacement Bst DNA polymerase, these primers bind to target DNA and form specific “loop” structures, completing target amplification under constant temperature (60–65 °C).

Primer design represents the greatest technical challenge in LAMP detection. Below are its core design principles:

1. Primer Components

Outer Primers (F3/B3): 1 primer each; core function is to initiate the strand-displacement reaction.
Inner Primers (FIP/BIP): 1 primer each; contain complementary sequences and are critical for loop structure formation.
Loop Primers (LF/LB, optional): Accelerate the amplification reaction and improve detection sensitivity.

2. Target Sequence Selection

Prefer sequence regions of 150–300 bp.
Choose highly conserved sequences with no cross-reactivity.
Avoid regions prone to intramolecular secondary structures.

3. Key Parameters for Primer Design

Distance Between Primer Regions

Distance between the 5′ end of F2 and the 5′ end of B2: 120–180 bp
Distance between the 5′ end of F2 and the 5′ end of F1c: ~40–60 bp
Distance between the 3′ end of F3 and the 5′ end of F2: ~0–60 bp
LoopF: located between the 3′ end of F2c and the 5′ end of F1c
LoopB: located between the 3′ end of B1 and the 5′ end of B2

Melting Temperature (Tm)

Tm values are adjusted based on base composition:

~60–65 °C for GC-rich or normal sequences
~55–60 °C for AT-rich sequences

Specific Tm standards:

F1c/B1c: 64–65 °C
F2/B2: 59–61 °C
F3/B3: 59–61 °C
LoopF/LoopB: 64–65 °C

Primer End Stability

Calculate the ΔG value for the last 6 bp of critical primer ends. The value must be < −4 kcal/mol. Applicable regions:

5′ end of F1c/B1c
3′ end of F2/B2
Ends of F3/B3

GC Content

GC-rich or normal sequences: 50–60%
AT-rich sequences: 40–50%

Secondary Structure

Primers must be designed to avoid self-forming secondary structures.

The 3′ end should not be AT-rich.
No complementary pairing with other primers.

Compared with qPCR, LAMP is more prone to false-positive results, mainly caused by primer dimers. We recommend testing multiple primer sets at different reaction temperatures to select the set with the best detection performance.