Fig. 3: Evaluating and enhancing the robustness of LAMP.

a Schematic of LAMP. Six distinct regions in the target locus (F1, F2, F3, B1, B2, and B3) are recognized by four core primers, which have a black arrow at their 3’ ends to represent extension by the DNA polymerase. FIP is represented by a dark green rectangle joined to a slanted light blue rectangle. BIP is represented by a dark brown rectangle joined to a slanted light purple rectangle. In addition, the F3 displacement primer is represented by a red rectangle, while the B3 displacement primer is represented by an orange rectangle. The letter “c” appended to each region name indicates the reverse complementary sequence. After our LAMP optimization process, we incorporated swarm primers, whose sequences are equivalent to F1c and B1c. Moreover, to demonstrate how a mismatch at the 5’ end of FIP can affect the LAMP reaction, we have added a yellow asterisk to track the progression of the mismatch. b Sequences of LAMP primers tested. The mismatches are indicated by bold red letters. c Strip chart showing how mismatches between LAMP primers and their binding sites affected the rate of isothermal amplification. RT-LAMP was performed at 65 °C in a real-time instrument with 20,000 copies of synthetic RNA corresponding to the S-gene of SARS-CoV-2. Cycle-threshold (Ct) values were given by the instrument using default settings. The black horizontal bars among the data points in the strip chart represent the mean (n = 3 [F3 MM, B3 MM] or 4 [PM, FIP (3’) MM, BIP (3’) MM, FIP (5’) MM, BIP (5’) MM, NTC] biological replicates). P-values were calculated using one-sided Student’s t-test. d Strip chart showing rescue of the LAMP reaction by truncated primers and a Q5 high-fidelity DNA polymerase in the presence of mismatches at the 3’ ends of FIP and BIP. RT-LAMP was performed at 65 °C with 20,000 copies of RNA template. The black horizontal bars among the data points in the strip chart represent the mean (n = 4 [FIP PM + tPM+Q5, BIP PM + tPM+Q5], 5 [PM, PM + Q5, FIP MM + tPM+Q5, FIP PM + tPM, BIP MM + tPM+Q5, BIP PM + tPM], or 6 [FIP MM, FIP MM + Q5, FIP MM + tPM, BIP MM, BIP MM + Q5, BIP MM + tPM, NTC] biological replicates). P-values were calculated using one-sided Student’s t-test. e Strip chart showing rescue of the LAMP reaction by truncated primers and a Q5 high-fidelity DNA polymerase in the presence of a mismatch at the 5’ end of FIP. RT-LAMP was performed at 65 °C with 20,000 copies of RNA template. The black horizontal bars among the data points in the strip chart represent the mean (n = 4 biological replicates). P-values were calculated using one-sided Student’s t-test. Source data are available in the Source Data file.