A pyrosequencing protocol for rapid identification of SARS-CoV-2 variants.

Next-generation sequencing (NGS) is the primary method used to monitor the distribution and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants around the world; however, it is costly and time-consuming to perform and is not widely available in low-resourced geographical regions. Pyrosequencing has the potential to augment surveillance efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. The current study describes the development of a reverse transcription (RT)-PCR-pyrosequencing assay targeting >65 spike protein gene (S) mutations of SARS-CoV-2, which permits differentiation of commonly reported variants currently circulating in the United States with a high degree of confidence. Variants typed using the assay included B.1.1.7 (Alpha), B.1.1.529 (Omicron), B.1.351 (Beta), B.1.375, B.1.427/429 (Epsilon), B.1.525 (Eta), B.1.526.1 (Iota), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), P1 (Gamma), and B.1.1 variants, all of which were confirmed by the NGS data. An electronic typing tool was developed to aid in the identification of variants based on mutations detected by pyrosequencing. The assay could provide an important typing tool for rapid identification of candidate patients for monoclonal antibody therapies and a method to supplement SARS-CoV-2 surveillance efforts by identification of circulating variants and novel emerging lineages.

Evaluation of transport media for laboratory detection of SARS-CoV-2 in upper respiratory tract swab specimens.

The reduced availability of commercial swabs and transport media for testing and administrative demands for increased testing capacity during the coronavirus disease 2019 (COVID-19) public health emergency has seriously challenged national laboratory testing programs, forcing many to use nontraditional collection devices, often without typical analytical assessment of their suitability in testing. Five common transport media (four commercial and one in-house) were evaluated for their suitability in the collection of nasopharyngeal swab specimens for subsequent molecular detection of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2). Results suggest that these transport media provide dependable temporal stability of the SARS-CoV-2 virus without significant analytical interference of molecular assays. These findings are not only important for addressing critical laboratory supply chain shortages of transport media in the current COVID-19 health crisis but also for future pandemic planning, when again supplies of commercially available transport media might be depleted.