Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

Abstract

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure.

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

“…The total assay time for RT-LAMP was 40 minutes, which is similar to that reported by Xu et al [7] but faster than what was reported by Kurosaki et al [26]. Furthermore, the LOD of this assay for direct detection of EBOV particles spiked into blood samples was 2.8 × 10 2 PFU/test for ZEBOV-Kikwit and 1 × 10 3 PFU/test for EBOV-Makona.…”

“…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.…”

“…The cost of the assay is also higher because of the addition of lateral flow cassettes. Kurosaki et al [26] developed an RT-LAMP method performed on the Genie II instrument (Optigene, United Kingdom), in which the readout is based on detection of turbidity. This method is suitable for use with only extracted RNA and cannot be used to detect EBOV directly from blood samples, as the color of blood will make it difficult to detect the turbidity, making it unstable for POC situations.…”

Maternal and Child Health Services in the Context of the Ebola Virus Disease: Health Care Workers’ Knowledge, Attitudes and Practices in Rural Guinea

“…Only recently, promising results of a rapid EVD diagnostic test (RDT) evaluated in Guinea have been published 19 . If approved, the availability of a RDT at rural health facilities would considerably facilitate the screening of suspected patients in maternal and child health services and reduce unnecessary referrals.…”

Biological Warfare Agents and their Detection and Monitoring Techniques (Review Paper)

“…LAMP assays have been used to detect various biological warfare agents including B. anthracis 35 , Y. pestis 36 , Brucella spp. 37 , B. pseudomallei 38 , C. burnetii 39 , Ebola virus 40 , Marburg virus 41 , and Lassa virus 42. Recombinase polymerase amplification (RPA) is another variant of DNA amplification techniques 43 . RPA does not employ thermal denaturation of template and works at a low and constant temperature (37 °C -40 °C).…”