Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection
Abstract 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 traditio...
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| Format: | Article |
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Nature Publishing Group
2025-07-01
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| Series: | Microsystems & Nanoengineering |
| Online Access: | https://doi.org/10.1038/s41378-025-00982-8 |
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| author | Kenia Chávez-Ramos Frida Trejo Prisciluis Caheri Salas-Navarrete Eva Ramón-Gallegos José Esteban Muñoz-Medina Luis Alvarez-Icaza Luis F. Olguin Oscar Pilloni Laura Oropeza-Ramos |
| author_facet | Kenia Chávez-Ramos Frida Trejo Prisciluis Caheri Salas-Navarrete Eva Ramón-Gallegos José Esteban Muñoz-Medina Luis Alvarez-Icaza Luis F. Olguin Oscar Pilloni Laura Oropeza-Ramos |
| author_sort | Kenia Chávez-Ramos |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-9a19252d95614f5fb79d4f8ce7d4f542 |
| institution | Kabale University |
| issn | 2055-7434 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Publishing Group |
| record_format | Article |
| series | Microsystems & Nanoengineering |
| spelling | doaj-art-9a19252d95614f5fb79d4f8ce7d4f5422025-08-20T03:45:49ZengNature Publishing GroupMicrosystems & Nanoengineering2055-74342025-07-0111111610.1038/s41378-025-00982-8Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detectionKenia Chávez-Ramos0Frida Trejo1Prisciluis Caheri Salas-Navarrete2Eva Ramón-Gallegos3José Esteban Muñoz-Medina4Luis Alvarez-Icaza5Luis F. Olguin6Oscar Pilloni7Laura Oropeza-Ramos8Instituto de Ingeniería, Universidad Nacional Autónoma de MéxicoInstituto de Ingeniería, Universidad Nacional Autónoma de MéxicoInstituto de Ingeniería, Universidad Nacional Autónoma de MéxicoLaboratorio de Citopatología Ambiental, ENCB, Instituto Politécnico Nacional (IPN), Campus Zacatenco, Calle Wilfrido Massieu Esquina Cda. Manuel Stampa, Col. Zacatenco. Alcaldia Gustavo A. MaderoDivisión de Laboratorios Especializados, Instituto Mexicano del Seguro SocialInstituto de Ingeniería, Universidad Nacional Autónoma de MéxicoLaboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de MéxicoInstituto de Ingeniería, Universidad Nacional Autónoma de MéxicoFacultad de Ingeniería, Universidad Nacional Autónoma de MéxicoAbstract 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.https://doi.org/10.1038/s41378-025-00982-8 |
| spellingShingle | Kenia Chávez-Ramos Frida Trejo Prisciluis Caheri Salas-Navarrete Eva Ramón-Gallegos José Esteban Muñoz-Medina Luis Alvarez-Icaza Luis F. Olguin Oscar Pilloni Laura Oropeza-Ramos Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection Microsystems & Nanoengineering |
| title | Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection |
| title_full | Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection |
| title_fullStr | Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection |
| title_full_unstemmed | Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection |
| title_short | Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection |
| title_sort | critical aspects of droplet digital reverse transcription loop mediated isothermal amplification ddrt lamp for viral pathogens detection |
| url | https://doi.org/10.1038/s41378-025-00982-8 |
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