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Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR.

ABSTRACT: The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.

SUBMITTER: Hong W

PROVIDER: S-EPMC8285242 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family: Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamoré (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe. In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/?L of RNA for RT-qPCR and 10 copies/?L of RNA for Semi-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses.

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Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR.

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