Development of an RT-LAMP assay for the detection of Lassa viruses in southeast and south-central Nigeria

Abstract

Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in 73 plasma collected from suspected LF patients admitted into two health settings in southern Nigeria. The clinical evaluation using the conventional RT-PCR as the reference test revealed a sensitivity of 50% in general with 100% for samples with a viral titer of 9500 genome equivalent copies (geq)/mL and higher. The detection limit was estimated to be 4214 geq/mL. The assay showed 98% specificity with no cross-reactivity to other viruses which cause similar symptoms. These results suggest that this RT-LAMP assay is a useful molecular diagnostic test for LF during the acute phase, contributing to early patient management, while using a convenient device for field deployment and in resource-poor settings.

Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification

“…Lassa virus (LASV) has 6 lineages and has highly diverse genome even within the same lineage. There is a single report of a RT-LAMP assay for LASV, and the assay requires 3 sets of primers to detect only lineage II [ 22 ]. Zika virus also shows diversity in genome sequences between Asian and African genotypes, requiring the mixture of 2 primer sets to detect all strains in one reaction [ 13 ].…”

Long-term validation of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 from March 2020 to October 2021 in Central Africa, Gabon

“…The advantages of the assay include diagnoses of infectious diseases on-site or in rural areas where resources and stable electricity are largely limited. Before the emergence of COVID-19, we had developed a variety of RT-LAMP assays to detect viruses that are a concern to public health, such as Ebola virus, Marburg virus, Lassa virus, and Zika virus [18][19][20][32][33][34][35]. In particular, RT-LAMP assays targeting Ebola and Zika viruses were tested on-site during the outbreaks, detecting target viruses within 30 min.…”

Potential and action mechanism of favipiravir as an antiviral against Junin virus

“…To quantify the encapsidated viral RNA copy numbers, the free RNA not associated with virions was removed from the samples using the Benzonase nuclease (Sigma), according to the manufacturer's instructions, prior to viral RNA extraction. Standard RNA was synthesized from a partial region of the GPC gene using the forward (5 0 -TAATACGACTCACTATAGG GCCAACCTTTTTGCAGGAGGC-3 0 ) and reverse (5 0 -AGCTTCTTCTGTGCAGGATCTT CCTGCAAGCGCTAGGAAT-3 0 ) primers and the T7 RNA polymerase (Promega), as previously described [69]. The prepared RNA was then serially diluted using DEPC-treated water to obtain a standard curve ranging from 10 2 -10 13 copies/mL.…”