Development and Characterization of Phage QBeta-Based SARS-CoV-2 RT-qPCR Assay Calibration Standards - Project Summary SARS-CoV-2 RT-PCR assays, one of the two major testing modalities used for diagnosis of COVID-19, are inherently quantitative. This has led to use of cycle threshold values for determining (a) if a tested individual remains infectious; (b) whether therapeutic approaches are working in immunocompromised patients; (c) whether a specific illness is more or less likely to be a result of SARS-CoV-2 infection or some other cause; and (d) whether a positive PCR test represents a current infection or prolonged detection of residual SARS-CoV-2 RNA that may linger for months after an original infection. Cycle threshold (Ct) values are the first cycle during PCR amplification during which fluorescence signal crosses a reliable instrument detection threshold. Unfortunately, Ct values are both assay and instrument specific. In fact, for any given Ct value, viral loads (the amount of virus in a nasal or nasopharyngeal sample) may vary over one thousand-fold depending on the testing platform. Traditionally, quantitative PCR assays are calibrated with standards which allow conversion of a test method-dependent Ct value to a test method-independent viral load. The latter directly reflects the amount of virus in a sample. Therefore, there is strong impetus to develop calibrators covering the billion-fold range of viral loads observed in COVID-19 infected individuals. Furthermore, these calibrators should be inexpensive and accessible to diagnostic and research laboratories. However, currently available calibration standards are very expensive, available in small quantities, labile, and/or do not reflect the higher viral load levels that identify infectious individuals and signify ongoing infection in immunocompromised patients. Therefore, here we propose to develop calibrators that address these needs. We identified the positive-strand RNA Qb bacteriophage as a compelling vector in which to clone SARS-CoV-2 PCR target sequence for calibrator development. Importantly, as an encapsidated single-strand RNA calibrator, such Qb derivatives will be able to assess inefficiencies in extraction, reverse transcription, and amplification phases of PCR tests; are inherently stable; and can be grown to high titers that encompass the upper range of viral loads observed in specimens. The proposal will examine the ability of the Qb to tolerate heterologous SARS-CoV-2 PCR target sequence inserted into Qb A1 and RNA- dependent RNA polymerase genes with or without complementation of phage functionality in trans. Lastly, we will validate calibrators through interrogation of two major commercial platform diagnostic SARS-CoV-2 PCR assays, and, as proof-of-principle, use these calibrators as a tool to establish their comparative analytical measurement range and limit of detection. Taken together, we will establish a new set of inexpensive, readily available reagents for use by the clinical and scientific community to enhance standardization and rigor of data available from SARS-CoV-2 PCR diagnostics.
