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Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2.
Michel, Janine; Neumann, Markus; Krause, Eva; Rinner, Thomas; Muzeniek, Therese; Grossegesse, Marica; Hille, Georg; Schwarz, Franziska; Puyskens, Andreas; Förster, Sophie; Biere, Barbara; Bourquain, Daniel; Domingo, Cristina; Brinkmann, Annika; Schaade, Lars; Schrick, Livia; Nitsche, Andreas.
  • Michel J; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Neumann M; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Krause E; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Rinner T; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Muzeniek T; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Grossegesse M; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Hille G; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Schwarz F; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Puyskens A; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Förster S; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Biere B; Influenza and Other Respiratory Viruses (FG 17), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Bourquain D; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Domingo C; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Brinkmann A; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Schaade L; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Schrick L; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
  • Nitsche A; Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany. NitscheA@rki.de.
Virol J ; 18(1): 110, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1255943
Semantic information from SemMedBD (by NLM)
1. Real-Time Polymerase Chain Reaction DIAGNOSES 2019 novel coronavirus
Subject
Real-Time Polymerase Chain Reaction
Predicate
DIAGNOSES
Object
2019 novel coronavirus
2. DNA Sequence PART_OF 2019 novel coronavirus
Subject
DNA Sequence
Predicate
PART_OF
Object
2019 novel coronavirus
3. Assay USES Detection
Subject
Assay
Predicate
USES
Object
Detection
4. Real-Time Polymerase Chain Reaction DIAGNOSES 2019 novel coronavirus
Subject
Real-Time Polymerase Chain Reaction
Predicate
DIAGNOSES
Object
2019 novel coronavirus
5. DNA Sequence PART_OF 2019 novel coronavirus
Subject
DNA Sequence
Predicate
PART_OF
Object
2019 novel coronavirus
6. Assay USES Detection
Subject
Assay
Predicate
USES
Object
Detection
ABSTRACT

BACKGROUND:

The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply.

AIM:

Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol).

METHODS:

Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen.

RESULTS:

Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits.

CONCLUSION:

The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.
Subject(s)
Keywords

Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Viral / Real-Time Polymerase Chain Reaction / COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Clinical Practice Guide Limits: Humans Language: English Journal: Virol J Journal subject: Virology Year: 2021 Document Type: Article Affiliation country: S12985-021-01559-3

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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Viral / Real-Time Polymerase Chain Reaction / COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Clinical Practice Guide Limits: Humans Language: English Journal: Virol J Journal subject: Virology Year: 2021 Document Type: Article Affiliation country: S12985-021-01559-3