Degenerate sequence-based CRISPR diagnostic for Crimean–Congo hemorrhagic fever virus
Fig 3
The degenerate sequence-based CRISPR diagnostic detects CCHFV in a sensitive, specific and rapid manner.
A–G. Detection of all CCHFV clades/sub-clades. The S segment genomic RNAs representing different CCHFV clades/sub-clades were produced by in vitro transcription, serially diluted and tested for detection by the degenerate CRISPR set (CRISPR set 2). Amplification plots show relative fluorescent units (RFU) at indicated time points in the T7-Cas13a reaction. RNA template concentrations in the T7-Cas13a reaction are shown as copies/μl (cp/μl). Red curves: CCHFV RNA-containing samples; green solid curve (0 cp/μl): NTC (no-template control, H2O); and green dash curve (Neg): the negative threshold = Mean + 3X standard deviation of NTCs, calculated based on 12 NTC replicates. Any RFU value above the negative threshold is considered CCHFV positive. Graphs represent three independent experiments showing similar patterns. H. Limit of detection (LoD). The S segment genomic RNA from the Hoti strain was produced by in vitro transcription and tested at the indicated concentrations for detection by the degenerate CRISPR set. Positivity% values are shown based on 10 replicates per target concentration type. I. The degenerate sequence-based CRISPR diagnostic is specific for CCHFV, with no cross-reactivity against closely related viruses. RNAs from CCHFV and related viruses were tested for detection by the degenerate CRISPR set. NSD: Nairobi sheep disease virus; RVFV: Rift Valley fever virus; and NTC: no-template control (H2O). CCHFV and Rift Valley fever virus RNAs were extracted from cell culture-derived viruses. Other viral RNAs were the S segment genomic RNAs produced by in vitro transcription. RNA template concentrations in the T7-Cas13a reaction were all 40 pg/μl. Amplification plots show relative fluorescent units (RFU) at indicated time points in the T7-Cas13a reaction and represent three independent experiments showing similar patterns.