Simultaneous rapid detection of Hantaan virus and Seoul virus using RT-LAMP in rats.

Abstract

BackgroundHemorrhagic fever with renal syndrome is in most cases caused by the Hantaan virus (HTNV) and Seoul virus (SEOV). To develop and apply reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect HTNV and SEOV simultaneously, which was faster, more cost effective, and easier to perform as the target gene amplified rapidly. In this article an assay based on LAMP is demonstrated, which only employs such apparatus as a water bath or a heat block.MethodsA chromogenic method using the calcein/Mn2+ complex and real-time turbidity monitoring method were used to assess reaction progress of the reaction, and the specificity of the RT-LAMP-based assay was assessed by detecting cDNAs/cRNAs generated from Coxsackievirus A16, Influenza virus, lymphocytic choriomeningitis virus, mouse poxvirus, rotavirus, mouse hepatitis virus. In addition, 23 clinical specimens were used to determine the agreement between the RT-LAMP assay with reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence (IFT) method.ResultsThe detection limit of RT-LAMP to HNTV and SEOV was as low as 10 copies/μL with optimized reaction conditions, which was much more sensitive than the RT-PCR method (100–1,000 copies/μL). At the same time, the detection results of 23 clinical specimens have also illustrated the agreement between this the RT-LAMP assay with RT-PCR and IFT.DiscussionThis RT-LAMP assay could be used to perform simultaneous and rapid detection of HTNV and SEOV to the clinical specimens.