One-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Nyaruaba, Raphael; Li, Xiohong; Mwaliko, Caroline; Ogolla, Faith; Li, Changchang; Zhao, Lu; Yang, Hang; Yu, Junping; Wei, Honping

doi:10.1007/978-1-0716-2111-0_10

“…Samples were then transported to the MP4 screening lab where they were immediately placed in a 65°C water bath for 35 minutes, previously shown to be effective for inactivation of SARS-CoV-2 53,54 . After inactivation, sample barcodes were scanned and 64 samples were organized into an 8 x 8 matrix whose placement was maintained when transferring to a 96-well microtiter sample plate (SP).…”

Section: Saliva Collectionmentioning

confidence: 99%

“…Droplets were then read on a QX200 Droplet Reader (BioRad) set up to read FAM and HEX channels. For additional details on the Bio-Rad RT-ddPCR work ow refer to the protocol by Nyaruaba et al 54 .…”

Section: Reverse Transcription Droplet Digital Pcr (Rt-ddpcr)mentioning

confidence: 99%

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

Wagner

¹

,

Fox

²

,

Gordon

³

et al. 2023

Preprint

0

View full text Add to dashboard Cite

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-hour turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

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“…Samples were then transported to the MP4 screening lab where they were immediately placed in a 65°C water bath for 35 minutes, previously shown to be effective for inactivation of SARS-CoV-2 49,50 . After inactivation, sample barcodes were scanned and 64 samples were organized into an 8 x 8 matrix whose placement was maintained when transferring to a 96-well microtiter sample plate (SP).…”

Section: Saliva Collectionmentioning

confidence: 99%

“…Droplets were then read on a QX200 Droplet Reader (BioRad) set up to read FAM and HEX channels. For additional details on the Bio-Rad RT-ddPCR work ow refer to the protocol by Nyaruaba et al 50 .…”

Section: Reverse Transcription Droplet Digital Pcr (Rt-ddpcr)mentioning

confidence: 99%

See 1 more Smart Citation

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

Wagner

¹

,

Fox

²

,

Gordon

³

et al. 2022

Preprint

0

View full text Add to dashboard Cite

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-hour turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

show abstract

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

Wagner

¹

,

Fox

²

,

Gordon

³

et al. 2023

Sci Rep

View full text Add to dashboard Cite

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

show abstract

One-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Cited by 3 publications

References 16 publications

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva

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