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J Biomed Nanotechnol. 2019 Apr 1;15(4):790-798. doi: 10.1166/jbn.2019.2742.
CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection.
Liu Y, Xu H, Liu C, Peng L, Khan H, Cui L, Huang R, Wu C, Shen S, Wang S, Liang W, Li Z, Xu B, He N.
Abstract
It is urgent to find an avian influenza A H7N9 detection simple method which is suitable for on-site detection. The Cas13a protein just likes a nanomachine, when specifically bound to target RNA by single-stranded RNA (crRNA), changes its protein structure and produces RNase activity, which degrades RNA non-specifically. Harnessing Cas13a, the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method. LwCas13a protein nanomachine was expressed in a prokaryotic expression system and purified by nickel column. In vitro transcribed RNA of H7N9 HA gene has been used as a target, to design a specific crRNA. The activity of Cas13a was verified with a single-stranded RNA-bound fluorescent group and a quenching fluorophore as signals. Using Cas13a, a room temperature H7N9 detection system was established. Detection of 1 nm of single-stranded RNA can be done within 5 min. When combined with the RT-RPA and T7 transcription system at room temperature, the detection limits of HA and NA are 1 fM and the reaction time is 50 min. Excellent specificity was achieved by comparison with subtype viruses such as H1N1 and H5N1. The rapid detection method based on CRISPR-Cas13a nanomachine H7N9 has been successfully established, which can detect H7N9 quickly and specifically. In the future, it can be quickly detected in the field with portable fluorescence detector.
PMID: 30841971 DOI: 10.1166/jbn.2019.2742
CRISPR-Cas13a Nanomachine Based Simple Technology for Avian Influenza A (H7N9) Virus On-Site Detection.
Liu Y, Xu H, Liu C, Peng L, Khan H, Cui L, Huang R, Wu C, Shen S, Wang S, Liang W, Li Z, Xu B, He N.
Abstract
It is urgent to find an avian influenza A H7N9 detection simple method which is suitable for on-site detection. The Cas13a protein just likes a nanomachine, when specifically bound to target RNA by single-stranded RNA (crRNA), changes its protein structure and produces RNase activity, which degrades RNA non-specifically. Harnessing Cas13a, the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method. LwCas13a protein nanomachine was expressed in a prokaryotic expression system and purified by nickel column. In vitro transcribed RNA of H7N9 HA gene has been used as a target, to design a specific crRNA. The activity of Cas13a was verified with a single-stranded RNA-bound fluorescent group and a quenching fluorophore as signals. Using Cas13a, a room temperature H7N9 detection system was established. Detection of 1 nm of single-stranded RNA can be done within 5 min. When combined with the RT-RPA and T7 transcription system at room temperature, the detection limits of HA and NA are 1 fM and the reaction time is 50 min. Excellent specificity was achieved by comparison with subtype viruses such as H1N1 and H5N1. The rapid detection method based on CRISPR-Cas13a nanomachine H7N9 has been successfully established, which can detect H7N9 quickly and specifically. In the future, it can be quickly detected in the field with portable fluorescence detector.
PMID: 30841971 DOI: 10.1166/jbn.2019.2742