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Principal Investigator: JAMES E KIRBY
Organization: BETH ISRAEL DEACONESS MEDICAL CENTER
Fiscal Year: 2024
Award: $216,125
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><Address><Assay><Bacteriophages><Bioassay><Biological Assay><COVID diagnosis><COVID diagnostic><COVID-19><COVID-19 diagnosis><COVID-19 diagnostic><COVID-19 infection><COVID-19 virus><COVID-19 virus infection><COVID19 diagnosis><COVID19 diagnostic><COVID19 infection><COVID19 virus><CV-19><Calibration><Capsid><Cell Communication and Signaling><Cell Signaling><Characteristics><Clinical><CoV-2><CoV2><Code><Coding System><Communities><Complement><Complement Proteins><Coronavirus Infectious Disease 2019><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Data><Decision Making><Detection><Development><Diagnostic><EC 2.7.7.48><Engineering><Fluorescence><Functional RNA><Gene Transcription><Genes><Genetic Transcription><Genome><Genome Stability><Genomic Stability><Goals><Hybrids><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><Individual><Infection><International><Intracellular Communication and Signaling><Investigators><Laboratory Research><Location><Measurement><Methods><Modality><Nasal><Nasal Passages Nose><Nasopharynx><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Nose><Patients><Phages><Phase><Plasmids><Polymerase Gene><Production><Public Health><RNA><RNA Bacteriophages><RNA Expression><RNA Gene Products><RNA Phages><RNA Polymerases><RNA Replicase><RNA-Dependent RNA Polymerase><RNA-Directed RNA Polymerase><RT-PCR><RTPCR><Reagent><Recombinants><Research Personnel><Research Specimen><Researchers><Residual><Residual state><Respiratory System, Nose, Nasal Passages><Reverse Transcriptase Polymerase Chain Reaction><Reverse Transcription><Rhinopharynx><Ribonucleic Acid><SARS><SARS corona virus 2><SARS coronavirus disease><SARS-CO-V2><SARS-COVID-2><SARS-CoV disease><SARS-CoV-2><SARS-CoV-2 diagnosis><SARS-CoV-2 diagnostic><SARS-CoV-2 infection><SARS-CoV2><SARS-CoV2 infection><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sampling><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome coronavirus disease><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Specimen><Standardization><System><T7 RNA polymerase><Testing><Therapeutic><Transcription><Ultrafiltration><Untranslated RNA><Viral Burden><Viral Load><Viral Load result><Virus><Wuhan coronavirus><bacterial virus><biological signal transduction><clinical care><clinical decision-making><comparative><complementation><coronavirus diagnosis><coronavirus diagnostic><coronavirus disease 2019><coronavirus disease 2019 diagnosis><coronavirus disease 2019 diagnostic><coronavirus disease 2019 infection><coronavirus disease 2019 virus><coronavirus disease diagnosis><coronavirus disease diagnostic><coronavirus disease-19><coronavirus disease-19 virus><coronavirus infectious disease-19><detection assay><detection limit><developmental><diagnosed with COVID><diagnosed with COVID-19><diagnosed with COVID19><diagnosed with SARS-CoV-2><diagnosed with coronavirus><diagnosed with coronavirus 2019><diagnosed with coronavirus disease><diagnosed with coronavirus disease 2019><diagnostic platform><diagnostic system><digital><genomic RNA><hCoV19><immunosuppressed patient><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><instrument><nCoV2><nasopharnygeal><noncoding><research study><reverse transcriptase PCR><severe acute respiratory syndrome coronavirus 2 diagnosis><severe acute respiratory syndrome coronavirus 2 diagnostic><tool><vector>