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.

中文翻译：

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基于CRISPR-Cas13a纳米机的简单技术用于禽流感（H7N9）病毒现场检测。

迫切需要找到一种适用于现场检测的简单的A7型禽流感H9N9检测方法。Cas13a蛋白就像一个纳米机器，当通过单链RNA（crRNA）与目标RNA特异性结合时，会改变其蛋白结构并产生RNase活性，从而非特异性降解RNA。利用Cas13a，本文旨在建立一种潜在的现场H7N9病毒核酸检测方法。LwCas13a蛋白质纳米机器在原核表达系统中表达并通过镍柱纯化。体外H7N9 HA基因的转录RNA已被用作靶标，以设计特定的crRNA。用单链RNA结合的荧光基团和淬灭荧光团作为信号验证了Cas13a的活性。使用Cas13a，建立了室温H7N9检测系统。可以在5分钟内完成1 nm单链RNA的检测。在室温下与RT-RPA和T7转录系统结合使用时，HA和NA的检出限为1 fM，反应时间为50分钟。通过与亚型病毒（例如H1N1和H5N1）进行比较，获得了出色的特异性。成功建立了基于CRISPR-Cas13a纳米机H7N9的快速检测方法，可以快速，特异性地检测H7N9。将来，可以使用便携式荧光检测器在现场快速检测到它。