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Comparison of SARS-CoV-2 N gene real-time RT-PCR targets and commercially available mastermixes
- Source :
- Journal of Virological Methods
- Publication Year :
- 2021
- Publisher :
- Elsevier B.V., 2021.
-
Abstract
- Background This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000–1 copy/μl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. Results Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). Conclusions This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.
- Subjects :
- 0301 basic medicine
Serial dilution
Coronavirus disease 2019 (COVID-19)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
030106 microbiology
Biology
Nose
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity
Homology (biology)
Article
Mastermix
03 medical and health sciences
N gene
Virology
Sequence Homology, Nucleic Acid
Coronavirus Nucleocapsid Proteins
Humans
SARS-CoV-2 RT-PCR
DNA Primers
Reverse Transcriptase Polymerase Chain Reaction
SARS-CoV-2
RNA
COVID-19
Phosphoproteins
Molecular biology
Reverse transcriptase
030104 developmental biology
Real-time polymerase chain reaction
COVID-19 Nucleic Acid Testing
Pharynx
RNA, Viral
Reagent Kits, Diagnostic
Primer (molecular biology)
Subjects
Details
- Language :
- English
- ISSN :
- 18790984 and 01660934
- Volume :
- 295
- Database :
- OpenAIRE
- Journal :
- Journal of Virological Methods
- Accession number :
- edsair.doi.dedup.....499479e430cff826df738708a383ef9f