Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

Abstract

Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks

“…Their recommendations also include monitoring patients’ viral loads ( 150 ), as studies have associated lower infectivity with a decrease in the SARS-CoV-2 viral load ( 151 – 153 ). Since dPCR has the capability of absolute quantification, it has been used to monitor viral loads of patients and to monitor disease progression in COVID-19 patients during hospitalization and after discharge ( 23 , 69 , 76 , 129 , 147 , 154 ). For example, after analyzing the viral loads in various clinical samples, Yu et al ( 69 ) discovered that the viral loads in the sputum samples of patients could reflect COVID-19 disease progression in vivo .…”

“…However, this method has not been widely used due to the limited sensitivity of RT-qPCR in testing samples with low viral loads. It has also been suggested that RT-dPCR may be more effective than RT-qPCR in pooled samples due to its higher sensitivity, specificity, and precision; same-day turnaround time; and relatively low cost ( 23 , 164 , 165 ). To test the suitability of RT-dPCR for pooling, Martin et al divided 448 COVID-19 hospital samples into three groups (14 pools of 32 samples/pool, 28 pools of 16 samples/pool, and 56 pools of 8 samples/pool) for RT-dPCR testing and directly compared the results to those of individual testing by RT-qPCR ( 165 ).…”

Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review

“…The studies were conducted worldwide; 16 in Asia [ 12 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ], 14 in Europe [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ], and 9 in North America [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ]. A minority of studies (8) included control samples [ 21 , 28 , 29 , 33 , 38 , 47 , 54 , 56 ]. One study did not report the number of samples or patients studied [ 26 ].…”

SARS-CoV-2 Viral Load Quantification, Clinical Findings and Outcomes in Children Seeking Emergency Department Care: Prospective Cohort Study

“…Samples were transported to an APL laboratory at room temperature and tested upon receipt using one of the following assays: BD MAX SARS-CoV-2 (BD Company, Sparks, MD), Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA), Seegene Allplex 2019-nCoV (Seegene Inc., Seoul, Korea) or the APL developed RT-PCR test (text, Supplemental Digital Content 1; http://links.lww.com/INF/E699 ). Archived samples, which were stored at −70°C, were tested by ddPCR using the validated 3 Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad, Pleasanton, CA, USA) (text, Supplemental Digital Content 2; http://links.lww.com/INF/E699 ).…”