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Comparative Study
. 2021 Sep:295:114215.
doi: 10.1016/j.jviromet.2021.114215. Epub 2021 Jun 21.

Comparison of SARS-CoV-2 N gene real-time RT-PCR targets and commercially available mastermixes

Julianne R Brown  1 , Denise M O'Sullivan  2 , Divya Shah  3 , Laura Atkinson  3 , Rui P A Pereira  2 , Alexandra S Whale  2 , Eloise J Busby  2 , Jim F Huggett  4 , Kathryn Harris  3
Affiliations
Comparative Study

Comparison of SARS-CoV-2 N gene real-time RT-PCR targets and commercially available mastermixes

Julianne R Brown et al. J Virol Methods. 2021 Sep.

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.

Keywords: Mastermix; N gene; SARS-CoV-2 RT-PCR.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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