Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR

Abstract

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.

Keywords: PMA; PMAxx; SARS-CoV-2; ddPCR; digital droplet PCR; negative-chain PCR; propidium monoazide; viral isolation; virus infectivity; virus integrity.

Figure 1

ddPCR results of serial dilutions of SARS-CoV-2 (live and UV-inactivated), both untreated and treated with 50 µM of PMAxx. NT: not treated.

Figure 2

(A) ddPCR results of 40 NT and treated with 50 µM of PMAxx NPSs from SARS-CoV-2-positive patients; (B) ddPCR results and mean of NT (red spots) and treated with 50 µM of PMAxx (green spots) NPSs from SARS-CoV-2-positive patients with Ct ≤ 25; (C) ddPCR results of NT (red spots) and treated with 50 µM of PMAxx (green spots) NPSs from SARS-CoV-2-positive patients with Ct > 25. For graphical representation and statistical analysis, an arbitrary value of −1.0 Log copies/mL was assigned to negative samples. NT: not treated. ****: p < 0.0001.

Figure 3

Negative-chain PCR and ddPCR results of six NPSs. For graphical representation, an arbitrary value of −1.0 Log copies/mL was assigned to negative samples. NT: not treated.