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Comparison of SARS-CoV-2 N gene real-time RT-PCR targets and commercially available mastermixes.
Brown, Julianne R; O'Sullivan, Denise M; Shah, Divya; Atkinson, Laura; Pereira, Rui P A; Whale, Alexandra S; Busby, Eloise J; Huggett, Jim F; Harris, Kathryn.
  • Brown JR; Microbiology, Virology and Infection Prevention and Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom. Electronic address: julianne.brown@gosh.nhs.uk.
  • O'Sullivan DM; National Measurement Laboratory at LGC, Teddington, United Kingdom.
  • Shah D; Microbiology, Virology and Infection Prevention and Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom.
  • Atkinson L; Microbiology, Virology and Infection Prevention and Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom.
  • Pereira RPA; National Measurement Laboratory at LGC, Teddington, United Kingdom.
  • Whale AS; National Measurement Laboratory at LGC, Teddington, United Kingdom.
  • Busby EJ; National Measurement Laboratory at LGC, Teddington, United Kingdom.
  • Huggett JF; National Measurement Laboratory at LGC, Teddington, United Kingdom; School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, United Kingdom.
  • Harris K; Microbiology, Virology and Infection Prevention and Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom.
J Virol Methods ; 295: 114215, 2021 09.
Article in English | MEDLINE | ID: covidwho-1275556
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Coronavirus Nucleocapsid Proteins / COVID-19 Nucleic Acid Testing / SARS-CoV-2 Type of study: Diagnostic study / Experimental Studies Limits: Humans Language: English Journal: J Virol Methods Year: 2021 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Coronavirus Nucleocapsid Proteins / COVID-19 Nucleic Acid Testing / SARS-CoV-2 Type of study: Diagnostic study / Experimental Studies Limits: Humans Language: English Journal: J Virol Methods Year: 2021 Document Type: Article