Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2

Kim, Kyoung Bo; Choi, Hayoung; Lee, Gun Dong; Lee, Jaewoong; Lee, Seungok; Kim, Yonggoo; Cho, Sung‐Yeon; Lee, Dong Gun; Kim, Myungshin

doi:10.1007/s40291-021-00547-1

Cited by 21 publications

(17 citation statements)

References 41 publications

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“…Close follow-up and systematic data collection allowed for determination of the role of viral load in transmission. We used the droplet digital PCR (ddPCR) technique to quantify SARS-CoV-2 viral RNA due to its greater accuracy and precision compared to traditional quantitative PCR (rRT-PCR) [15,16]. Moreover, we compared the SAR for the Alpha variant with the SAR for other circulating variants in Norway during the study period.…”

Section: Introductionmentioning

confidence: 99%

Household Transmission of SARS-CoV-2: A Prospective Longitudinal Study Showing Higher Viral Load and Increased Transmissibility of the Alpha Variant Compared to Previous Strains

et al. 2021

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We studied the secondary attack rate (SAR), risk factors, and precautionary practices of household transmission in a prospective, longitudinal study. We further compared transmission between the Alpha (B.1.1.7) variant and non-Variant of Concern (non-VOC) viruses. From May 2020 throughout April 2021, we recruited 70 confirmed COVID-19 cases with 146 household contacts. Participants donated biological samples eight times over 6 weeks and answered questionnaires. SARS-CoV-2 infection was detected by real-time RT-PCR. Whole genome sequencing and droplet digital PCR were used to establish virus variant and viral load. SARS-CoV-2 transmission occurred in 60% of the households, and the overall SAR for household contacts was 50%. The SAR was significantly higher for the Alpha variant (78%) compared with non-VOC viruses (43%) and was associated with a higher viral load. SAR was higher in household contacts aged ≥40 years (69%) than in younger contacts (40–47%), and for contacts of primary cases with loss of taste/smell. Children had lower viral loads and were more often asymptomatic than adults. Sleeping separately from the primary case reduced the risk of transmission. In conclusion, we found substantial household transmission, particularly for the Alpha variant. Precautionary practices seem to reduce SAR, but preventing household transmission may become difficult with more contagious variants, depending on vaccine use and effectiveness.

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Section: Introductionmentioning

confidence: 99%

Household Transmission of SARS-CoV-2: A Prospective Longitudinal Study Showing Higher Viral Load and Increased Transmissibility of the Alpha Variant Compared to Previous Strains

et al. 2021

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“…An overall of 3651 samples, from 2825 patients and 145 controls, were extracted from the 39 included studies. 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 ].…”

Section: Resultsmentioning

confidence: 99%

“…Evidence suggests that ddPCR assays exhibit an enhanced analytical sensitivity, as assessed by measuring their limit of detection (LOD). Abasiyanik et al, reported LODs of 0.06 and 0.21 copies/µL, while Kim et al 1.99 (95% CI: 1.38–3.38) and 5.18 copies/µL (95% CI: 3.33–12.93), respectively, while targeting the N1 and N2 genes [ 32 , 48 ]. Two other groups designed their ddPCR assays targeting the ORF1ab and N genes; Dong et al measured LODs of 2 copies/reaction for both genes, whereas Nyaruaba et al 1.42 and 2.75 copies/reaction for the ORF1ab and N genes, respectively [ 22 , 27 ].…”

Section: Resultsmentioning

confidence: 99%

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

Ishak

AlRawashdeh

Esagian

et al. 2021

JCM

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Accurate detection of SARS-CoV-2, the pathogen causing the global pandemic of COVID-19, is essential for disease surveillance and control. Quantitative reverse transcription PCR (RT-qPCR) is considered the reference standard test for the diagnosis of SARS-CoV-2 by the World Health Organization and Centers for Disease Control and Prevention. However, its limitations are a prompt for a more accurate assay to detect SARS-CoV-2, quantify its levels, and assess the prognosis. This article aimed to systematically review the literature and assess the diagnostic performance of droplet digital PCR (ddPCR), also to evaluate its potential role in prognosis and management of COVID-19 patients. PubMed and Scopus databases were searched to identify relevant articles published until 13 July 2021. An additional PubMed search was performed on 21 October 2021. Data from the 39 eligible studies were extracted and an overall 3651 samples from 2825 patients and 145 controls were used for our qualitative analysis. Most studies reported ddPCR was more accurate than RT-qPCR in detecting and quantifying SARS-CoV-2 levels, especially in patients with low viral loads. ddPCR was also found highly effective in quantifying SARS-CoV-2 RNAemia levels in hospitalized patients, monitoring their disease course, and predicting their response to therapy. These findings suggest ddPCR could serve as a complement or alternative SARS-CoV-2 tool with emerging diagnostic, prognostic, and therapeutic value, especially in hospital settings. Additional research is still needed to standardize its laboratory protocols, also to accurately assess its role in monitoring COVID-19 therapy response and in identifying SARS-CoV-2 emerging variants.

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“…Kappa ( K ) value is interpreted as poor ( K < 0.00), slight (0.00 < K < 0.20), fair (0.20 < K < 0.40), moderate (0.41 < K < 0.60), substantial (0.61 < K < 0.80), and almost perfect (0.81 < K < 1.00). In addition, we performed the dilutional linearity experiments by spike-in the high concentration of COVID-19 positive sample (Ct value near 10) to the negative sample to compare the detection capability according to the dilution factor (10 −1 to 10 −10 ) of two kits (Kim et al 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Diagnostic evaluation of qRT-PCR-based kit and dPCR-based kit for COVID-19

et al. 2021

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Background Coronavirus disease of 2019 (COVID-19) is well known as a fatal disease, first discovered at Wuhan in China, ranging from mild to death, such as shortness of breath and fever. Early diagnosis of COVID-19 is a crucial point in preventing global prevalence. Objective We aimed to evaluate the diagnostic competency and efficiency with the Allplex™ 2019-nCoV Assay kit and the Dr. PCR 20 K COVID-19 Detection kit, designed based on the qRT-PCR and dPCR technologies, respectively. Methods A total of 30 negative and 20 COVID-19 positive specimens were assigned to the diagnostic test by using different COVID-19 diagnosis kits. Diagnostic accuracy was measured by statistical testing with sensitivity, specificity, and co-efficiency calculations. Results Comparing both diagnostic kits, we confirmed that the diagnostic results of 30 negative and 20 positive cases were the same pre-diagnostic results. The diagnostic statistics test results were perfectly matched with value (1). Cohen’s Kappa coefficient was demonstrated that the given kits in two different ways were “almost perfect” with value (1). In evaluating the detection capability, the dilutional linearity experiments substantiate that the Dr. PCR 20 K COVID-19 Detection kit could detect SARS-CoV-2 viral load at a concentration ten times lower than that of the Allplex™ 2019-nCoV Assay kit. Conclusions In this study, we propose that the dPCR diagnosis using LOAA dPCR could be a powerful method for COVID-19 point-of-care tests requiring immediate diagnosis in a limited time and space through the advantages of relatively low sample concentration and small equipment size compared to conventional qRT-PCR. Supplementary Information The online version contains supplementary material available at 10.1007/s13258-021-01162-4.

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Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2

Cited by 21 publications

References 41 publications

Household Transmission of SARS-CoV-2: A Prospective Longitudinal Study Showing Higher Viral Load and Increased Transmissibility of the Alpha Variant Compared to Previous Strains

Household Transmission of SARS-CoV-2: A Prospective Longitudinal Study Showing Higher Viral Load and Increased Transmissibility of the Alpha Variant Compared to Previous Strains

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

Diagnostic evaluation of qRT-PCR-based kit and dPCR-based kit for COVID-19

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