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 pat...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^(R)as the fluorescent dye was crucial,as other dyes tested diffused into the oil phase.Optimal amplification occurred with 105μm 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.展开更多
基金preliminary LAMP experiments in bulk by Dr.Elizabeth Castillo-Villanueva.K.C.R.acknowledges Secretaría de Ciencia,Humanidades,Tecnología e Innovación(SECIHTI),for a postdoctoral scholarship(CVU 662463)supported by UNAM-PAPIIT grants IT100922 and IN211623,SECTEI grant SECTEI/248/2021the Faculty of Chemistry,UNAM(PAIP 5000-9023).
文摘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^(R)as the fluorescent dye was crucial,as other dyes tested diffused into the oil phase.Optimal amplification occurred with 105μm 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.
