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Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7.
Nörz, Dominik; Grunwald, Moritz; Olearo, Flaminia; Fischer, Nicole; Aepfelbacher, Martin; Pfefferle, Susanne; Lütgehetmann, Marc.
  • Nörz D; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
  • Grunwald M; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
  • Olearo F; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
  • Fischer N; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
  • Aepfelbacher M; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
  • Pfefferle S; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
  • Lütgehetmann M; Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany. Electronic address: mluetgeh@uke.de.
J Clin Virol ; 141: 104894, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1267739
Preprint
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ABSTRACT

BACKGROUND:

New SARS-CoV-2 variants with increased transmissibility, like B.1.1.7, first detected in England or B.1.351, first detected in South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand.

METHODS:

A set of RT-qPCR assays was modified for a diagnostic SARS-CoV-2 multiplex assay including detection of the del-HV69/70 and N501Y mutations on the cobas6800 platform. Analytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. For clinical performance, a total of 176 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and next generation sequencing as gold standard.

RESULTS:

The multiplex assay was highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 6.16 cp/ml (CI 4.00-8.31). LoDs were slightly higher for detection of the HV69/70 deletion (85.92, CI 61-194.41) and the N501Y SNP (105.99 cp/ml, CI 81.59 - 183.66). A total of 176 clinical samples were tested with the assay, including 50 samples containing SARS-CoV-2 of the B.1.1.7 lineage, one containing B.1.351 and 85 non-B.1.1.7/B.1.351 lineage, of which three also harbored a HV69/70 deletion. All were correctly identified by the multiplex assay.

CONCLUSION:

We describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of the del-HV69/70 and N501Y mutations that can distinguish between B.1.1.7 and other lineages. The assay allows for high-throughput screening for currently relevant variants in clinical samples prior to sequencing.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study Topics: Variants Limits: Humans Language: English Journal: J Clin Virol Journal subject: Virology Year: 2021 Document Type: Article Affiliation country: J.jcv.2021.104894

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study Topics: Variants Limits: Humans Language: English Journal: J Clin Virol Journal subject: Virology Year: 2021 Document Type: Article Affiliation country: J.jcv.2021.104894