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We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM–1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies. [ABSTRACT FROM AUTHOR]

This study provides proof of concept of a colorimetric biosensor for influenza H1N1 virus assay based on the CRISPR/Cas13a system and hybridization chain reaction (HCR). Target RNA of influenza H1N1 virus activated the trans-cleavage activity of Cas13a, which cleaved the special RNA sequence (-UUU-) of the probe, further initiating HCR to copiously generate G-rich DNA. Abundant G-quadruplex/hemin was formed in the presence of hemin, thus catalyzing a colorimetric reaction. The colorimetric biosensor exhibited a linear relationship from 10 pM to 100 nM. The detection limit was 0.152 pM. The biosensor specificity was excellent. This new and sensitive detection method for influenza virus is a promising rapid influenza diagnostic test. [ABSTRACT FROM AUTHOR]