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Yale IMPACT Research Team,

Publications and source records attributed to Yale IMPACT Research Team,.

2 recordsLinked to original sources

Detection of long SARS-CoV-2 nucleocapsid sequences in peripheral blood monocytes collected soon after hospital admission

Many viruses infect circulating mononuclear cells thereby facilitating infection of diverse organs. Blood monocytes (PBMC) are being intensively studied as immunologic and pathologic responders to the new SARS-CoV-2 virus (CoV19) but direct evidence showing CoV19 in monocytes is lacking. Circulating myeloid cells that take up residence in various organs can harbor viral genomes for many years in lymphatic tissues and brain, and act as a source for re-infection and/or post-viral organ pathology. Because nucleocapsid (NC) proteins protect the viral genome we tested PBMC from acutely ill patients for the diagnostic 72bp NC RNA plus adjacent longer (301bp) transcripts. In 2/11 patient PBMC, but no uninfected controls, long NCs were positive as early as 2-6 days after hospital admission as validated by sequencing. Pathogenic viral fragments, or the infectious virus, are probably disseminated by rare myeloid migratory cells that incorporate CoV19 by several pathways. Predictably, these cells carried CoV19 to heart and brain educing the late post-viral pathologies now evident.

microbiology

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR

The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the CDC, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.

microbiology