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

Abstract

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

“…Using ddPCR, it has been shown that SARS-CoV-2 could reliably be detected from nasopharyngeal samples without RNA extraction [18,19]. In our flowchart ( Figure 8A), we also highlight this as a possibility even though we did not explore it.…”

“…Since the discovery and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan, China [1], the virus has spread to multiple countries across the globe causing a global public health crisis. Many health systems have been crippled by this pandemic and all efforts are geared towards prevention, diagnosis, and treatment of the virus and its associated disease coronavirus disease 2019 (COVID- 19).…”

Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing

“…Using ddPCR, it has been shown that SARS-CoV-2 could reliably be detected from nasopharyngeal samples without RNA extraction [ 22 , 23 ]. In our flowchart ( Figure 8A ), we also highlight this as a possibility even though we did not explore it.…”

Point of Care Diagnostics in the Age of COVID-19

“…Moreover, Deiana and et al described a simple approach for SARS-CoV-2 viral load quantification in nasopharyngeal swab using direct ddPCR without the need for RNA extraction and purification. They successfully reported the detection of mostly equal copies of RNA in the direct compared to the extracted approach, with at least 80% sensitivity and 93% specificity [59]. Besides all the advantages of these RT-PCR techniques, given all the requirements for sample preparation and long turn-around time, this method is severely limited in handling pandemic situations, particularly in cities with a fast-growing number of infected patients.…”