Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection
Por:
Chávez-Ramos K., Trejo F., Salas-Navarrete P.C., Ramón-Gallegos E., Muñoz-Medina J.E., Alvarez-Icaza L., Olguin L.F., Pilloni O., Oropeza-Ramos L.
Publicada:
1 ene 2025
Resumen:
The COVID-19 pandemic evidenced the urgent need for rapid, accurate, and scalable diagnostic methods for emerging infectious diseases. Droplet digital reverse transcription LAMP (ddRT-LAMP) is a promising technique for pathogen detection and accurate quantification, as it overcomes traditional LAMP’s limitations in viral load estimation through reaction partitioning and digital analysis. However, many parameters must be adjusted to avoid spurious results. This study evaluates the critical conditions for effective ddRT-LAMP quantification of the SARS-CoV-2 N gene in plasmid DNA, synthetic RNA, and nasopharyngeal swab samples. Using a polydimethylsiloxane (PDMS) microfluidic device, the RT-LAMP reaction mixture with a fluorescent dye was divided into thousands of droplets stabilized by a surfactant in fluorinated oil. After incubation, the droplets were injected into a PDMS chamber for fluorescent imaging to determine the proportion of positive droplets and quantify the samples based on the Poisson distribution. The results showed that primer design and master mix composition significantly impacted the amplification. The selection of GelGreen® as the fluorescent dye was crucial, as other dyes tested diffused into the oil phase. Optimal amplification occurred with 105 µm droplet diameter and 30-min incubation, achieving detection and quantification limits of 102 cp/µL. By addressing these operational challenges, ddRT-LAMP can become a more effective tool for viral detection and quantification in clinical diagnostics. (Figure presented.) © The Author(s) 2025.
Filiaciones:
Instituto de Ingeniería, Universidad Nacional Autónoma de México, Mexico City, 04510, Mexico
Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, 04510, Mexico
Facultad de Ingenieria, Universidad Nacional Autonoma de Mexico, Mexico City, 04510, Mexico
Laboratorio de Citopatología Ambiental, ENCB, Instituto Politécnico Nacional (IPN), Campus Zacatenco, Calle Wilfrido Massieu Esquina Cda. Manuel Stampa, Col. Zacatenco. Alcaldia Gustavo A. Madero, Mexico City, 07738, Mexico
División de Laboratorios Especializados, Instituto Mexicano del Seguro Social, Mexico City, 07760, Mexico
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