How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

Autor: Anantharajah A; Department of Microbiology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium. Electronic address: ahalieyah.anantharajah@uclouvain.be., Helaers R; Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels, Belgium., Defour JP; Department of Hematology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium; Ludwig Institute for Cancer Research & de Duve Institute, Université catholique de Louvain, Brussels, Belgium., Olive N; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Kabera F; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Croonen L; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Deldime F; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Vaerman JL; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Barbée C; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Bodéus M; Department of Microbiology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Scohy A; Department of Microbiology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Verroken A; Department of Microbiology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Rodriguez-Villalobos H; Department of Microbiology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium., Kabamba-Mukadi B; Department of Microbiology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium; Department of Molecular Biology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2021 Sep; Vol. 295, pp. 114197. Date of Electronic Publication: 2021 May 24.
DOI: 10.1016/j.jviromet.2021.114197
Abstrakt: Objectives: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection.
Methods: We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID-19. An additional 30 "pre-intervention screening" samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets.
Results: The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives.
Conclusion: An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.
(Copyright © 2021 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: