Multifunctional single-hairpin mediated enzyme-cascade quadruple enrichment system coupled with CRISPR/Cas12a for ultrasensitive hantavirus detection

Hantavirus (HV) is widely distributed worldwide. It causes hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), which has a mortality rate of up to 15 % and 40 %. In this work, a single hairpin-based enzyme-cascade quadruple enrichment (SH-ECE) system coupled with CRISPR/ cas12a was designed as a sensing platform for the detection of a Orthohantavirus dabieshanense (DBSV). With the assistance of two enzymes, the SH-ECE system can be constructed by using only one hairpin probe (HP). When the target gene is present, the SH-ECE system will be activated, and four different cyclic reactions will occur sequentially. These reactions proceed continuously, realizing quadruple amplification and outputting a large number of mimic target DNA and functional strands, which will participate in the subsequent reactions. Impressively, the introduction of CRISPR/ cas12a technology further amplifies the signal while enhancing the specificity and sensitivity of the sensing platform, even enabling the discrimination of RNA with single-, double-, and triple-base mutations. The sensing platform can detect DBSV with a LOD of 16.7 fM (S/N = 3) for linearity spanning from 50 fM to 50 nM. The applicability of this method was validated using both human serum specimens and rodent serum samples. Based on this, the research on this sensing method can be widely applied to guide and facilitate the further development of CRISPR/Cas and SH-ECE system-based assays.