Biology subjects

Vargas-Reyes, M.

Publications and source records attributed to Vargas-Reyes, M..

3 recordsLinked to original sources

bioRxiv2026

A Portable Fluorescence Platform for Decentralized One-Health mcr-1 Monitoring

Antimicrobial resistance (AMR) represents a major global health threat, with plasmid-borne mcr genes driving colistin resistance and exposing critical gaps in One-Health surveillance across human, animal, and environmental reservoirs. The most prevalent variant, mcr-1, remains difficult to monitor in resource-limited settings due to the lack of rapid, affordable, and field-deployable molecular tools. Here, we developed C12amcr, an integrated molecular toolbox that combines pre-amplification PCR with a fluorescent CRISPR-Cas12a assay targeting a conserved region of mcr-1 and a custom low-cost, hand-held 3D-printed portable fluorometer. Under optimized conditions, the assay achieved a limit of detection of 630 cells/mL. In poultry feces spiked with mcr-1-positive E. coli, C12amcr detected as few as 1,800 cells/mL. When tested on 22 community-derived E. coli isolates, the assay showed 100% concordance with both next-generation sequencing for mcr-1 detection and phenotypic colistin susceptibility testing by broth microdilution. The accompanying portable fluorometer performed equivalently to a laboratory microplate reader while enabling fully decentralized workflows compatible with portable PCR platforms. By integrating locally produced molecular reagents, straightforward protocols, and an accessible field-ready fluorescence reader, C12amcr overcomes key barriers to decentralized AMR surveillance and provides a practical, scalable solution for One-Health monitoring in resource-limited settings.

molecular biology↗

bioRxiv2025

Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene

The {beta}-lactams are critically important broad-spectrum antibiotics, widely used as first-line treatments; however, their effectiveness is increasingly compromised by {beta}-lactamase enzymes. Among these, OXA-type enzymes have expanded to over 400 variants and are highly prevalent in Enterobacteriaceae. Current phenotypic and molecular detection tests have long turnaround times or require specialized equipment, respectively. In this study, we optimize a rapid molecular assay combining a PCR with modified thermal ramp rate (TRR) along with CRISPR-Cas12a fluorescence detection for the blaOXA-1gene. Using a commercial DNA Taq polymerase (TRR: 2.2 {degrees}C/s, annealing and extension hold time: 1 s), amplification time was reduced from 80 to 30 min, enabling detection within 50 min (PCR: 30 min; CRISPR: 20 min). With a locally produced enzyme (hold: 10 s), amplification time was 44 min. The assay achieved an analytical sensitivity of 8 CFU/reaction using commercial DNA Taq polymerase. The accelerated PCR:CRISPR workflow delivers results in less than one hour without compromising technical sensitivity (attomoles range), not requiring high technical expertise, and can be implemented in laboratories with basic molecular biology equipment. HighlightsAn optimized thermal gradient can reduce the turnaround time of PCR-based detection tests CRISPR-Cas in addition to the modified PCR can detect a gene target in less than an hour The proposed workflow is suitable for implementation with basic molecular biology equipment

molecular biology↗

bioRxiv2024

Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers

Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results redout (Fluorescence vs FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.

microbiology↗