A Universal Fluorescent Sensing System For Pathogen Determination Based On Loop-Mediated Isothermal Amplification Triggering Dual-Primer Rolling Circle Extension

The great challenge for loop-mediated isothermal amplification (LAMP) is target-specific detection. However, the most popular fluorogenic probe method needs complicated sequence design and large reagent consumption. Rolling circle amplification (RCA), known for its simplicity, is presumably compatible with LAMP in single-tube reaction detection based on Bst DNA polymerase to realize universality. Therefore, this work combined LAMP with RCA through strand displacement strategy to determine pathogens. Specifically, pathogen gene initiated LAMP process, displacing two single strands (S1 and S2) hybridized with LAMP loop primers for dual-primer rolling circle extension. Then, Molecular beacon (MB) probe paired to RCA amplicons emitting fluorescence signal for real-time detection. For detecting different pathogens, LAMP primers were specifically designed without changing the detection strategies through same RCA set. The established LAMP-RCA technique quantitatively detected invA and malB ranging from 10(2) to 10 (6) copies mu L-1, also demonstrating the universality. Meanwhile, S. typhimurium without genomic DNA extraction was directly quantified ranging from 10(2) to 3.16 x 10(4) CFU mu L-1, with a detection limit of 32 CFU mu L -1 . With good selectivity, this work was successfully applied for S. typhimurium determination in 10 % milk, indicating that the established method had considerable potential in complex samples.