TY - JOUR
T1 - Multiplex PCR Assays for Identifying all Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants
AU - Dikdan, Ryan J.
AU - Marras, Salvatore A.E.
AU - Field, Amanda P.
AU - Brownlee, Alicia
AU - Cironi, Alexander
AU - Hill, D. Ashley
AU - Tyagi, Sanjay
N1 - Publisher Copyright:
© 2022 Association for Molecular Pathology and American Society for Investigative Pathology
PY - 2022/4
Y1 - 2022/4
N2 - Variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including alpha, beta, gamma, delta, and omicron, threaten to prolong the pandemic, leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the evolution of the virus, but is costly, slow, and not easily accessible. Multiplex quantitative RT-PCR assays for SARS-CoV-2 have been developed that identify all VOCs as well as other mutations of interest in the viral genome, nine mutations in total, using single-nucleotide discriminating molecular beacons. The presented variant molecular beacon assays showed a limit of detection of 50 copies of viral RNA, with 100% specificity. Twenty-six SARS-CoV-2–positive patient samples were blinded and tested using a two-tube assay. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26 of 26). We have used our design methodology to rapidly design an assay that detects the new omicron variant. This omicron assay was used to accurately identify this variant in 17 of 33 additional patient samples. These quantitative RT-PCR assays identify all currently circulating VOCs of SARS-CoV-2, as well as other important mutations in the spike protein coding sequence. These assays can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.
AB - Variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including alpha, beta, gamma, delta, and omicron, threaten to prolong the pandemic, leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the evolution of the virus, but is costly, slow, and not easily accessible. Multiplex quantitative RT-PCR assays for SARS-CoV-2 have been developed that identify all VOCs as well as other mutations of interest in the viral genome, nine mutations in total, using single-nucleotide discriminating molecular beacons. The presented variant molecular beacon assays showed a limit of detection of 50 copies of viral RNA, with 100% specificity. Twenty-six SARS-CoV-2–positive patient samples were blinded and tested using a two-tube assay. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26 of 26). We have used our design methodology to rapidly design an assay that detects the new omicron variant. This omicron assay was used to accurately identify this variant in 17 of 33 additional patient samples. These quantitative RT-PCR assays identify all currently circulating VOCs of SARS-CoV-2, as well as other important mutations in the spike protein coding sequence. These assays can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.
UR - http://www.scopus.com/inward/record.url?scp=85127142644&partnerID=8YFLogxK
U2 - 10.1016/j.jmoldx.2022.01.004
DO - 10.1016/j.jmoldx.2022.01.004
M3 - Article
C2 - 35121139
AN - SCOPUS:85127142644
SN - 1525-1578
VL - 24
SP - 309
EP - 319
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 4
ER -