DNA amplification using RCA-LAMP

For PLP-mediated targeting, 1 µL of 10x reaction buffer, 2 µL of 10 nM PLP, and 5 µL of RNA were mixed. The mixture was incubated at 90 °C for 1 min, 70 °C for 1 min, and cooled to room temperature for 45 min. After that, 2 µL of Splint R ligase (6.75 U/µL) was added and incubated at 37 °C for 30 min for ligation, inactivated at 65 °C for 20 min and cooled at 4 °C.

For RCA-LAMP amplification, 1x reaction buffer, 1 mM each dNTP, 6 mM MgSO₄, 0.8 M betaine, 0.8 µM forward and reverse primer, 4U Bst 2.0 polymerase, and 1 µL of PLP ligation products were mixed and incubated at 68 °C for 3 h. The primers were replaced with rh primer, and 50 mU RNase H2 enzyme was added to run the rhPCR reaction for RCA-LAMP. Then, the DNA amplicons were analyzed using 2% agarose gel electrophoresis and stained with ethidium bromide.

Quick-Load Purple 1 kb Plus DNA Ladder (0.1. 10.0 kb) purchased from New England Biolabs (NEB; Massachusetts, USA) was used as the DNA ladder marker. Also, 1x EvaGreen was added to quantify DNA amplification in real-time. The fluorescence intensity of the RCA-LAMP reaction system was monitored in real-time using the Applied Biosystems Step One Plus real-time PCR system (Thermo Fisher Scientific) for 3 h at intervals of 1 min.