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

Qian Ying Wang; Fan Li; Xin Xin Shen; Shi Hong Fu; Ying He; Wen Wen Lei; Guo Dong Liang; Huan Yun Wang; Xue Jun Ma

doi:10.3967/bes2019.047

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

Biomed Environ Sci. 2019 May;32(5):357-362. doi: 10.3967/bes2019.047.

Authors

Qian Ying Wang¹, Fan Li¹, Xin Xin Shen², Shi Hong Fu¹, Ying He¹, Wen Wen Lei¹, Guo Dong Liang¹, Huan Yun Wang¹, Xue Jun Ma²

Affiliations

¹ Department of Viral Encephalitis, NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; State Key Laboratory for Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
² NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.

PMID: 31217052
DOI: 10.3967/bes2019.047

Abstract

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 °C 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.

Keywords: Detection; Far-eastern; RT-RAA; Subtype; Tick-borne encephalitis virus.

Publication types

Evaluation Study

MeSH terms

Encephalitis Viruses, Tick-Borne / genetics
Encephalitis Viruses, Tick-Borne / isolation & purification*
Nucleic Acid Amplification Techniques*
RNA, Viral / analysis*

Substances

RNA, Viral