Isothermal Real-Time RT-RPA for Machupo Virus Detection: Field-Adaptable Sensitivity Comparable with Laboratory PCR
Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Isothermal nucleic acid amplification methods, such as recombinase polymerase amplification (RPA), are becoming increasingly vital as diagnostic platforms for neglected tropical diseases (NTDs) by enabling rapid and accurate detection in under-resourced regions. Wse developed a real-time RT-RPA assay for Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever, and directly compared it to a real-time RT-PCR assay targeting the same viral sequence fragment. The methods were evaluated across critical parameters: limit of detection (LOD), tolerance to single-nucleotide substitutions, multiplexing capability, and adaptability to multiple MACV genetic variants. The LOD was identical for both assays: 5×10 3 copies/ml of armored RNA particles. They differed in terms of input RNA (copies/reaction): 100 (PCR) versus 20 (RPA). The real-time RT-RPA assay was further validated on a portable device, demonstrating its field-deployability for point-of-care applications.
Author Summary
We developed two new diagnostic tools to detect Machupo virus, which causes deadly Bolivian hemorrhagic fever in remote regions where lab resources are limited. Our tests, a lab-based RT-PCR and a portable rapid RT-RPA, both identify the virus with high sensitivity (detecting as few as 5,000 virus-mimicking particles per milliliter). The RT-RPA method works at a constant temperature, delivering results in 20–30 minutes on a handheld device, making it ideal for field clinics. While RT-PCR remains cost-effective ($1.15/test), RT-RPA ($5.95/test) offers unmatched speed and portability. Both tests perform well with viral genetic variability, ensuring reliable detection across strains. This work provides the first deployable molecular diagnostics tool for Machupo virus, filling a critical gap in neglected tropical disease outbreak response. Our study shows that rapid, field-ready diagnostics can match lab-based standards without sacrificing accuracy.
