medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Clarity Plus™ digital PCR: A novel platform for absolute quantification of SARS-CoV-2 Shawn Yi Han Tan2, Sheng Yi Milton Kwek1, Huiyu Low1, Yan Ling Joy Pang2 1JN Medsys, 217 Henderson Road #02-08, Singapore 159555, Singapore Institute of Technology, 10 Dover Dr, Singapore 138683, Singapore [email protected], [email protected], [email protected], [email protected] Abstract In recent years, the usage of digital polymerase chain reaction (dPCR) for various clinical applications has increased exponentially. Considering the growing demand for improved dPCR technology, the Clarity Plus™ dPCR system which features enhanced multiplexing capability and a wider dynamic range for nucleic acid analysis was recently launched. In this study, a dPCR assay optimized for use on Clarity PlusTM was evaluated for the absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for the global coronavirus disease 2019 (COVID-19) outbreak. The assay demonstrated good inter- and intraassay precision, accuracy, as well as excellent linearity across a range of over 6 orders of magnitude for target gene quantification. In addition, comparison of the assay on both dPCR and qPCR platforms revealed that dPCR exhibited a slightly higher sensitivity compared to its qPCR counterpart when quantifying SARS-CoV-2 at a lower concentration. Overall, the results showed that the dPCR assay is a reliable and effective approach for the absolute quantification of SARSCoV-2 and can potentially be adopted as a molecular tool for detecting low viral load in patients and wastewater surveillance of COVID-19. NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Keywords SARS-CoV-2 RT-dPCR assay, Absolute viral quantification, Clarity Plus™, Sample partitioning, Tube-strip digital PCR. 1. Introduction Digital Polymerase Chain Reaction (dPCR) was first introduced by Volgelstein and Kinzler in a 1999 publication in...

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . and the Naica® System (Stilla Technologies). The second method is a chip-based dPCR technology used by BioMark™ HD (Fluidigm), QuantStudio® 3D (Thermo Fischer Scientific), and Clarity™ (JN Medsys) [2, 6]. Since the mid-2000s, a plethora of new dPCR applications...

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . N2 sequences [12, 13]. Presently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of COVID-19 worldwide [14]. Various RT-qPCR assays have been developed worldwide which detect SARS-CoV-2...

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Using the Clarity™ auto loader, the mix was delivered onto the chip where it was sub-divided into 40,000 partitions and subsequently sealed with Clarity™ Seal Enhancer with addition of 245μl Clarity™ Sealing fluid (Fig 1). All sealed chips underwent another...

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . 2.3 SARS-CoV-2 linearity study and limit of quantification (LOQ) The Linearity and LOQ of SARS-CoV-2 RT-dPCR assay were obtained by quantifying a single copy of N1 and ORF1ab gene obtained from a series of serial dilutions with a range of expected target...

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . QuantStudio® 3 qPCR system is unable to read the Quasar670 fluorescence, only ORF1ab digital PCR data is provided. The extracted RNA stock was diluted 8 and 16 times to achieve an expected concentration of 0.225 and 0.1125 copies/μl respectively. Reagent...

medRxiv preprint doi: https://doi.org/10.1101/2021.05.30.21256718; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . 3. Results and Discussion 3.1 Absolute quantification of SARS-CoV-2 target using Clarity Plus™ system The linearity and the limit of quantification (LOQ) for the Clarity PlusTM SARS-CoV-2 RT-dPCR assay were first determined using N1 and ORF1ab Synthetic...