Loop mediated isothermal amplification (LAMP) is an effective quantitative technique that is widely used as a diagnostic tool and day-to-day research. LAMP paves way for fast and accurate detection of pathogens as it is a highly sensitive and specific method for point of care (POC) DNA amplification (1).

Nucleic acid amplification by LAMP consists of three steps: initiation, followed by cycling amplification, and finally elongation. As opposed to conventional polymerase chain reaction (PCR) which uses only two primers, amplification of a target gene by LAMP requires six primers: two inner primers (FIP and BIP), two outer primers (F3 and B3), and two loop primers (LF and LB). These primers work by creating a stem-loop structure with template DNA to produce initiation sites resulting in DNA amplification by a DNA polymerase with strand displacement (SD) and reverse transcriptase (RT) activities (Fig. 1). Polymerase SD activity is required to separate the two existing DNA strands while RT activity enables RNA targets to be detected (2).

Figure 1. LAMP primer mechanism for DNA amplification. Reference for drawing from New England Biolabs (3).

In terms of time consumption, sensitivity, and accuracy, LAMP stands well above conventional PCR and most other PCR techniques. Conventional PCR is more time consuming since multiple temperature cycles are required for each amplification step whereas LAMP is isothermal, meaning it requires only a single optimal temperature of 65°C (2). As a result, LAMP typically takes less than 60 minutes to complete. Amplification by LAMP is 10-fold to 100-fold more sensitive than conventional PCR methods (4). LAMP permits extremely specific amplification as all six primers are needed to correctly recognize their target site for amplification to proceed, consequently leading way to more accurate results (1). Overall, LAMP is well studied and is proven to have outstanding potential to provide quick and sensitive results for the detection of pathogens than other PCR techniques.

The gold standard DNA polymerase used in LAMP is Bst polymerase, a homologue of Bacillus stearothermophilus DNA polymerase I large fragment (5). Although Bst polymerase allows for rapid and sensitive DNA amplification, it lacks 5’ à 3’ exonuclease activity (5). This provides more room for error in DNA amplification which is certainly undesirable for LAMP amplification. In light of this, we introduced three single-point mutations to Bst polymerase to improve thermostability. Additionally, previous research indicates that fusion of a DNA-binding protein to a DNA polymerase is likely to enhance processivity of the LAMP reaction. In hopes of improving strand displacement activity and processivity of Bst polymerase, Sac7e, a double-stranded DNA-binding protein isolated from Sulfolobus acidocaldarius was fused to the N-terminus of Bst polymerase via a (GGGGS)4 flexible linker to create a newly improved Bst polymerase.