# Isothermal real-time RT-RPA for Machupo virus detection: Field-adaptable sensitivity comparable with laboratory PCR

## TL;DR

A new isothermal test for Machupo virus matches PCR sensitivity and can be used in the field for quick diagnosis.

## Contribution

A real-time RT-RPA assay for Machupo virus with field-deployable potential and sensitivity comparable to PCR.

## Key findings

- The RT-RPA and RT-PCR assays had the same limit of detection (5 × 103 copies/ml).
- RT-RPA required fewer RNA copies per reaction (20) compared to PCR (100).
- RT-RPA was validated on a portable device, showing promise for field use.

## Abstract

Isothermal nucleic acid amplification methods, such as recombinase polymerase amplification (RPA), are becoming increasingly vital as diagnostic platforms for neglected tropical diseases by enabling rapid and accurate detection in under-resourced regions. We 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 × 103 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 potential for field-deployability for point-of-care applications.