Related Experiment Video
Updated: Jan 15, 2026
DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning
Published on: May 10, 2024
CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of
Marina A Kapitonova1, Anna V Shabalina1, Vladimir G Dedkov1,2
1Laboratory of Pathogen Molecular Genetics, St. Petersburg Pasteur Institute, St. Petersburg 197101, Russia.
Abstract:
Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 104 copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.
Insights
A new RT-RPA/DETECTR assay offers rapid, equipment-free detection of Bolivian hemorrhagic fever (BHF) virus. This isothermal method accurately identifies multiple genetic variants of the Machupo virus (MACV) in clinical samples.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Bolivian hemorrhagic fever (BHF) is a severe zoonotic disease caused by the Machupo virus (MACV).
- Current diagnostic methods like RT-PCR require extensive laboratory infrastructure, limiting their use in endemic areas.
- There is a critical need for rapid, sensitive, and equipment-free diagnostic tools for BHF.
Purpose of the Study:
- To develop and validate a novel isothermal nucleic acid detection assay for MACV.
- To enable simultaneous detection of MACV genetic variants using a multiplex approach.
- To provide an accessible diagnostic alternative for resource-limited settings.
Main Methods:
- Development of a one-tube, isothermal assay combining recombinase polymerase amplification (RPA) with the Cas12a-based DETECTR system (RT-RPA/DETECTR).
- Optimization of the assay using engineered virus-like armored particles containing MACV target sequences.
- Validation of the multiplex assay using spiked clinical samples to assess sensitivity, specificity, and variant detection capabilities.
Main Results:
- The multiplex RT-RPA/DETECTR assay achieved a limit of detection of approximately 5 × 10^4 copies/mL (80 aM) of armored particles.
- The assay demonstrated the ability to simultaneously detect MACV genetic variants, including those with point mutations.
- Validation with clinical samples confirmed the assay's selectivity and reliability in detecting specific nucleotide substitutions.
Conclusions:
- The RT-RPA/DETECTR assay provides a sensitive, specific, and equipment-free method for MACV detection.
- This multiplex assay facilitates the simultaneous identification of diverse MACV strains, improving diagnostic capabilities.
- The developed assay represents a significant advancement for BHF diagnostics in resource-limited and endemic regions.
