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 different viruses. The aim of this study was to compare the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance on the direct quantitation of SARS-CoV-2 on nasopharyngeal swab respect to the procedure applied to the extracted RNA. Moreover, the two widely used swab types were compared (UTM 3mL and ESwab 1mL, COPAN). A total of 50 nasopharyngeal swabs (n=25 UTM 3mL and n=25 ESwab 1mL) from SARS-CoV-2 patients collected during the pandemic from 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 in order to perform 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 RNA extracted. 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 simple and fast approach for the 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 as we used thawed material and to the heating treatment. 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.…”

On-demand Ferrofluid Droplet Formation With Non-linear Magnetic Permeability in the Presence of a Non-uniform Magnetic Field

“…Therefore, Equations ( 1) and ( 2) can be modified as Equation (5). Magnetic permeability is a property of a substance that can be denoted by 𝜇 = 𝜇 0 (1 + 𝜒), in which 𝜒 is the magnetic susceptibility.…”

“…The dynamics of droplet formation has been investigated numerically, experimentally, and theoretically in different geometries including nozzles [1], and lab-on-a-chip systems [2]. Droplet generation has been utilized in various applications, such as 3D printing [3], electrosprays [4], and bioengineering [5]. Droplet formation is performed by passive or active methods, which differ in the absence or presence of an external force.…”

On-demand ferrofluid droplet formation with non-linear magnetic permeability in the presence of high non-uniform magnetic fields

“…The dynamics of droplet formation has been investigated numerically, experimentally, and theoretically in different geometries, including nozzles 1 and lab-on-a-chip systems 2 . Droplet generation has been utilized in various applications, such as 3D printing 3 , electrosprays 4 , and bioengineering 5 . Droplet formation is performed by passive 6 or active 7 methods, which differ in the absence or presence of an external force.…”