Sensitive one-step isothermal detection of pathogen-derived RNAs

Chang Ha Woo; Sungho Jang; Giyoung Shin; Gyoo Yeol Jung; Jeong Wook Lee

To increase sensitivity, current nucleic acid detection methods generally involve a 38 target amplification step prior to the detection step. The conventional amplification method is 39 based on PCR, which requires a thermocycler for delicate temperature modulation. As an 40 alternative to the thermal cycling-based amplification, isothermal amplification methods are 41 available, which rely primarily on a strand-displacing polymerase or T7 RNA polymerase at a 42 constant temperature 7 . However, the complex composition of the isothermal amplification 43 mixtures often renders these approaches incompatible with detection methods and whole 44 diagnosis generally becomes a multi-step process [8] [9] [10] [11] . The diagnostic regimen with multi-step 45 procedures requires additional time, instruments, and reagents, as well as skilled personnel to 46 perform the diagnostic procedure. This aspect limits the broad applicability of nucleic acid 47 diagnostics, especially in situations where rapid and simple detection is required. 48 3 efficiently ligate two DNA probes using a target single-stranded RNA as a splint, enabling 54 the sequence-specific detection of RNA molecule 17, 18 . Because the reaction components of 55 the ligation-dependent methods are relatively simple, we hypothesized that the ligation-56 dependent method could be exploited to establish a one-step RNA detection platform when 57 combined with compatible amplification and signal generation methods in a single reaction 58 mixture. 59