ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens

Abstract

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR

“…As of today, few studies reported the use of ddPCR for a more sensitive SARS-CoV-2 detection compared to RT-PCR 12 , 14 – 16 . To our knowledge, this is the first report of direct quantitation of SARS-CoV-2 RNA performed on a consistent number of clinical samples and comparing two different nasopharyngeal swabs.…”

“…In this context, the droplet digital PCR (ddPCR) might be more appropriate for quantitation of viral loads, as previously reported 7 . The ddPCR allows precise quantitation of nucleic acid copies without the need of a calibration curve and with higher resistance to the amplification inhibitors, compared to the quantitative real-time PCR 8 ; some recent studies reported the usage of ddPCR for the quantitation of SARS-CoV-2 9 – 16 . However, all the described ddPCR procedures included a RNA extraction/purification step, leading to potential amplification errors, due to variable and suboptimal nucleic acid yields 17 , 18 .…”

COVID‐19 diagnostic testing: Technology perspective

“…An overall accuracy of 94.3% was obtained, ∼500 times more sensitive than RT-PCR. 68 Lu et al compared the RT-dPCR and RT-PCR with 108 specimens (pharyngeal swab, stool, and blood) longitudinally collected from 36 COVID-19 patients. The RT-dPCR showed the detection limit 10× lower than that of RT-PCR.…”

Sensitive Detection and Quantification of SARS-CoV-2 by Multiplex Droplet Digital RT-PCR

“…While reverse-transcriptase RT-PCR is still the gold standard, the ndings in the present study indicate that the assay sensitivity of the RT-PCR assay is reduced due to background NA from the patient sample. By contrast, the sensitivity of the RT-ddPCR multiplex assay was not affected by background NA, and is more sensitive than the gold standard reverse-transcriptase RT-PCR [4][5][6][7] in the clinical setting.…”

“…Another approach is to detect the nucleocapsid (N) gene, and to use an open reading frame 1a/b (ORF1b) gene or E gene assay as a con rmatory test [3]. In addition, to improve assay sensitivity, other studies have been focusing on the detection of N, E or ORF1b using droplet digital polymerase chain reaction (ddPCR) [4][5][6][7].…”