A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus

摘要

Objective Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. Methods A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 ℃ within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. Results The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. Conclusion A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.