Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts

Autor: Joseph K. Wong, Ryan Marczak, Albert Vallejo-Gracia, G. Renuka Kumar, Sulggi A. Lee, Holly Anne Martin, Melanie Ott, Steven A. Yukl, Parinaz Fozouni, Sushama Telwatte, Victoria Murray
Jazyk: angličtina
Rok vydání: 2021
Předmět:
replication
Viral pathogenesis
viruses
Messenger
Clinical Sciences
Computational biology
Subgenomic RNA
Biology
medicine.disease_cause
Polymerase Chain Reaction
Viral transcription
General Biochemistry
Genetics and Molecular Biology
Article
03 medical and health sciences
Transcription (biology)
Genetics
medicine
Humans
Digital polymerase chain reaction
Viral
Guide RNA
RNA
Messenger
skin and connective tissue diseases
Lung
Molecular Biology
030304 developmental biology
Subgenomic mRNA
Coronavirus
0303 health sciences
SARS-CoV-2
Prevention
030302 biochemistry & molecular biology
fungi
RNA
virus diseases
COVID-19
Pneumonia
Reverse Transcription
Viral transcription/replication
Reverse transcriptase
body regions
Infectious Diseases
Droplet digital PCR
Quantitative assays
RNA
Viral
Digital PCR
Biotechnology
Zdroj: Methods (San Diego, Calif.)
ISSN: 1095-9130
1046-2023
Popis: The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.
Databáze: OpenAIRE
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