Platinum chloride-based viability RT-qPCR for SARS-CoV-2 detection in complex samples

Abstract

Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.

A Propidium Monoazide (PMAxx)-Droplet Digital PCR (ddPCR) for the Detection of Viable Burkholderia cepacia Complex in Nuclease-Free Water and Antiseptics

“…PMA can only penetrate bacterial cells with damaged membranes and covalently bind to double-stranded DNA upon exposure to bright visible light resulting in the amplification of only viable cells [ 21 ]. Successful use of the PMA assay has been reported on Escherichia coli , Salmonella enterica , Campylobacter coli , Brucella suis , and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in food and water [ 19 , 22 , 23 , 24 , 25 ]. PMAxx, an enhanced version of PMA was introduced in 2015 and showed better discriminative power than PMA between viable and dead cells [ 26 ].…”

Combined kinetic analysis of SARS-CoV-2 RNAemia, N-antigenemia and virus-specific antibodies in critically ill adult COVID-19 patients

“…Using a previously published protocol 24 , plasma specimens were diluted 1/10 in PBS, pretreated or not at high temperature (95 °C, 10 min) then treated with 5 mM of platinum chloride (PtCl 4 ) for 30 min at room temperature in DNA LoBind tubes (Eppendorf, Germany) in an orbital shaker (150 rpm). Viral RNA was then extracted using Maxwell ® RSC 16 instrument and Maxwell RSC Pure Food GMO and authentication kit (Promega, Spain) and amplified by RT-qPCR targeting the N gene (N1 sequence).…”

Recent findings and applications of biomedical engineering for COVID-19 diagnosis: a critical review

“…Modifications to the qRT-PCR procedure have been proposed and demonstrated to improve the overall capacity, reduce turn-around time, cost, or adapt the system to POCT settings, such as employing patient-collected swabs and saline gargles [ 107 ] or saliva [ 108 ,, 109 ], unextracted clinical samples [ 110–114 ], or portable miniature PCR workstations [ 115–117 ]. Noticeably, a novel approach to PCR, namely viability RT-qPCR, employed platinum chloride to treat NP swab samples and prevent the amplification of SARS-CoV-2 RNA in free form or from virions with damaged capsids, thus detecting the only RNA associated with intact virions, indicating infectivity [ 118 ]. This method is a suitable tool to ascertain one’s infectiousness without the need to perform virus culture, avoid false positives caused by contaminated RNA from the environment and identify noninfectious, prolonged RNA shedding from patients.…”