Critical aspects of droplet digital reverse transcription loop-mediated isothermal amplification (ddRT-LAMP) for viral pathogens detection.

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 10