Survey and Visual Detection of Zaire ebolavirus in Clinical Samples Targeting the Nucleoprotein Gene in Sierra Leone

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.

Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood

“…The sensitivity of our RT-LAMP method was found to be 10 RNA copies/µL, which is similar to that reported by others [7,24], and the specificity was high. This RT-LAMP method showed sensitivity similar to that of the real-time RT-PCR method for both strains of EBOV (Makona and Kikwit), indicating that this method can detect the low copy numbers of target RNA.…”

“…RT-LAMP methods for EBOV detection have been previously reported [7,[24][25][26], but all these methods require extraction of RNA from blood samples. Because the reaction developed in the present study is performed at high temperature, lysis and Figure 1.…”

A flexible format LAMP assay for rapid detection of Ebola virus

“…The colour assay showed additional sensitivity with good detection down to 50 copies and partial results at 20 copies (Fig 2). Although these may not be as sensitive as some already published RT-PCR assays for Ebola virus [36], they nevertheless show detection well within the clinical range [34,[37][38][39] and are comparable to other published isothermal assays with limits of detection commonly within the range of 50-500 copies of target [26,[40][41][42][43][44] and showing a significant improvement in limit of detection to the recently published Ebola virus RPA [44].…”

“…There are many examples in the literature of endpoint LAMP assays with visual detection, such as detection of the pyrophosphate biproduct of the amplification by turbidity or through colour change using a calcein dye, and detection of Mg 2+ ions with hydroxy napthol blue (HNB) dye, however, the results with these assays have been mixed [41,42,54]. Turbidity detection assays tend to have limited sensitivity with "by-eye" detection.…”

Evaluation on the diagnostic efficiency of different methods in detecting COVID-19

“…This method has high sensitivity, high specificity, simple method, low cost and time saving, which has been widely applied for the detection of influenza virus, MERS-CoV, West Nile virus, Ebola virus, Zika virus, yellow fever virus, and a variety of other pathogens (59)(60)(61)(62)(63)(64).…”