Detection of new bunyavirus RNA by reverse transcription-loop-mediated isothermal amplification

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging and epidemic infectious disease in central and northeast China. It is caused by New Bunyavirus and carries an average 12% case fatality rate. Early and rapid detection is critical for prevention and control of New Bunyavirus infection, since no vaccine or antiviral drugs are currently available, and prevention requires careful attention to control of the suspected tick vector. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of New Bunyavirus. The detection limit of the RT-LAMP assay was approximately 10(3) 50% tissue culture infective doses/ml of New Bunyavirus in culture supernatants, and no cross-reactive amplification of other viruses known to cause similar clinical manifestations was observed. The assay was further evaluated using 138 specimens from clinically suspected SFTS and 40 laboratory-proven hantavirus infection with fever and renal syndrome patients, and the assay exhibited 97% agreement compared to real-time RT-PCR and conventional RT-PCR. Using real-time RT-PCR as the diagnostic gold standard, RT-LAMP was 99% sensitive and 100% specific. The RT-LAMP assay could become a useful alternative in clinical diagnosis of SFTS caused by New Bunyavirus, especially in resource-limited hospitals or rural clinics of China.

FIG 1

RT-LAMP products detected by visual observation (A), UV light (B), and agarose gel electrophoresis (C). Lane 1, negative control; lanes 2 to 4, different New Bunyavirus strains; lanes 5 to 7, positive samples; lanes 8 to 10, negative samples; lane 11, positive control; lane M, DL2000 molecular size marker.

FIG 2

Detection sensitivity of RT-LAMP, RT-PCR, and real-time RT-PCR for New Bunyavirus. RT-LAMP products from virus serial dilutions were visualized by adding FDR dye under visible light (A), UV light (B), and by agarose gel electrophoresis (C). RT-PCR products were evaluated by agarose gel electrophoresis (D) and real-time RT-PCR amplification (E). Lane 1, negative control; lanes 2 to 8, NBV samples from 10¹ to 10⁷ TCID₅₀/ml serial dilutions; lane 9, positive control; lane M, DL2000 molecular size marker.