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

ABSTRACT: Background:Hemorrhagic 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. Methods:A 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. Results:The detection limit of RT-LAMP to HNTV and SEOV was as low as 10 copies/?L with optimized reaction conditions, which was much more sensitive than the RT-PCR method (100-1,000 copies/?L). 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. Discussion:This RT-LAMP assay could be used to perform simultaneous and rapid detection of HTNV and SEOV to the clinical specimens.

SUBMITTER: Sui X

PROVIDER: S-EPMC6329334 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Sui Xin X Zhang Xu X Fei Dongliang D Zhang Zhen Z Ma Mingxiao M

PeerJ 20190108

<h4>Background</h4>Hemorrhagic 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.<h4>Methods</h ...[more]

PMID: 30643674

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Project description:Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent "bloom" events for positive samples, in an approach called "Spatial LAMP" (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17-32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 102 copies per μl, and a gamma-irradiated virus of 103 virus particles in a single 12.5 μl droplet blood sample.

Dataset Information

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

Publications

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

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