Rapid and portable bunyavirus SFTSV RNA testing utilizing catalytic hairpin assembly coupled with lateral flow immunoassay

Abstract

ABSTRACT Severe fever with thrombocytopenia syndrome (SFTS) is a prevalent, life-threatening, emergent infectious disease. Currently, reverse transcription-polymerase chain reaction is the gold standard for diagnosing SFTS virus (SFTSV) infection, which requires sophisticated equipment and professional personnel that are frequently unavailable in most SFTS endemic rural areas. Here, we reported a simple, rapid nucleic acid amplification system that combined the catalytic hairpin assembly (CHA) with a lateral flow immunoassay (LFIA) strip-based detection method for SFTSV detection. The detection of SFTSV RNA could be realized by generation of H1-H2 hybrid duplexes labeled with biotin and digoxin, which subsequently added to the LFIA test strips containing streptavidin conjugated with Alexa Fluor 647 as well as anti-digoxin antibodies. Our CHA-based LFIA assay offered high amplification efficiency and specificity with a detection limit of 1 aM. Crucially, this method enabled stable detection of 500 copies/mL of SFTSV within 30 min using clinical serum samples. Therefore, our CHA-based LFIA approach provided a potential useful tool to facilitate early and precise diagnosis of SFTS patients in poorly resourced SFTS endemic areas. Importance Severe fever with thrombocytopenia syndrome (SFTS) is an emerging and potentially fatal infectious disease prevalent in China. Here we report a simple, rapid nucleic acid amplification system, the catalytic hairpin assembly (CHA) in conjunction with a lateral flow immunoassay (LFIA) strip-based detection method for SFTS virus detection, which demonstrated high amplification efficiency and specificity with limit of detection of 1 aM. Most importantly, we also validate our CHA-based LFIA assay using the clinical serum samples, which was fully compatible with reverse transcription-PCR results. Therefore, our strategy provides a potential useful tool to facilitate early and precise diagnosis of SFTS patients especially in poorly resourced SFTS endemic areas.

Keywords

• SFTSV
• catalytic hairpin assembly
• lateral flow immunoassay
• POCT