Development of simple and rapid assay to detect viral RNA of tick-borne encephalitis virus by reverse transcription-loop-mediated isothermal amplification

Daisuke Hayasaka,Kouichi Morita,Kotaro Aoki

doi:10.1186/1743-422x-10-68

Abstract

BackgroundTick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study.MethodsPrimers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change.ResultsThe sensitivity of TBEV-specific RT-LAMP assay was 102 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV.ConclusionTBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients.

Highlights

Tick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans
Our TBEV-specific RTLAMP assay was more sensitive for the transcribed RNA of the Oshima strain than the real-time RT-PCR (rRT-PCR) assay with TBEV-specific primers reported previously [14]
TBEV with the Hochosterwitz showing early amplification than the other strains (Figure 2E). These results indicate that our reverse-transcriptase loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assay can amplify the target RNA of all TBEV subtypes as the rRT-PCR assay established previously [14]

Summary

Introduction

Tick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. TBEV is transmitted by Ixodes tick species and rodents in nature, and infects humans through the bite of an infected tick [1,2,4] It is distributed over a wide area of Europe and Asia, and is geographically and genetically divided into three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) [5,6,7]. Surveillance on the prevalence of TBEV in ticks or serological monitoring of rodent is required to assess the risk of human infection caused by this virus in the endemic areas [9,10,11]

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