Abstract
Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. To guide treatment and prevent spread of the viral infection, a rapid and sensitive detection method is required for clinical samples. Here, we described and evaluated a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect Zaire ebolavirus using the nucleoprotein gene (NP) as a target sequence. Two different techniques were used, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring. The RT-LAMP assay detected the NP target sequence with a limit of 4.56 copies/μL within 45 min under 61°C, a similar even or increase in sensitivity than that of real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, all pseudoviral particles or non- Zaire EBOV genomes were negative for LAMP detection, indicating that the assay was highly specific for EBOV. To appraise the availability of the RT-LAMP method for use in clinical diagnosis of EBOV, of 417 blood or swab samples collected from patients with clinically suspected infections in Sierra Leone, 307 were identified for RT-LAMP-based surveillance of EBOV. Therefore, the highly specific and sensitive RT-LAMP method allows the rapid detection of EBOV, and is a suitable tool for clinical screening, diagnosis, and primary quarantine purposes.
Highlights
The 2014 Ebola virus (EBOV) outbreak was the largest to date in West African countries (Frieden et al, 2014; Hampton, 2014)
We developed a point-of-care reverse transcription loop-mediated isothermal amplification (RT-Loop-mediated isothermal amplification (LAMP)) assay targeted to nucleoprotein gene (NP)
reverse transcription-polymerase chain reaction (RT-PCR), and green fluorescence was observed after ∼45 min by RT-LAMP, indicating a low level of EOBV RNA
Summary
The 2014 Ebola virus (EBOV) outbreak was the largest to date in West African countries (Frieden et al, 2014; Hampton, 2014). It spread through direct contact with infected individuals, or by touching the blood, organs, bodily secretions and fluids, or the contaminated clothes of such people (MacNeil and Rollin, 2012). Inhibitors present in crude biological samples can inactivate the Taq DNA polymerase used in PCR-based techniques (de Franchis et al, 1988) These methods are relatively complex and require specialized instruments. To complement PCR-based methods, another rapid, simple, and effective assay is needed
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