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, G; Schwärz, F.; Puyskens, Andreas; Förster, Sophie; Biere, Barbara; Bourquain, Daniel; Domingo, Cristina; Brinkmann, Annika; Schaade, Lars; Schrick, Livia; Nitsche, Andreas

doi:10.1186/s12985-021-01559-3

Cited by 46 publications

(46 citation statements)

References 19 publications

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“…There is convincing evidence that infectivity of SARS-CoV-2 correlates directly with high viral loads in respiratory specimens of acutely infected persons [11][12][13].…”

Section: Discussionmentioning

confidence: 99%

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

et al. 2021

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Introduction Numerous CE-marked SARS-CoV-2 antigen rapid diagnostic tests (Ag RDT) are offered in Europe, several of them with unconfirmed quality claims. Aim We performed an independent head-to-head evaluation of the sensitivity of SARS-CoV-2 Ag RDT offered in Germany. Methods We addressed the sensitivity of 122 Ag RDT in direct comparison using a common evaluation panel comprised of 50 specimens. Minimum sensitivity of 75% for panel specimens with a PCR quantification cycle (Cq) ≤ 25 was used to identify Ag RDT eligible for reimbursement in the German healthcare system. Results The sensitivity of different SARS-CoV-2 Ag RDT varied over a wide range. The sensitivity limit of 75% for panel members with Cq ≤ 25 was met by 96 of the 122 tests evaluated; 26 tests exhibited lower sensitivity, few of which failed completely. Some RDT exhibited high sensitivity, e.g. 97.5 % for Cq < 30. Conclusions This comparative evaluation succeeded in distinguishing less sensitive from better performing Ag RDT. Most of the evaluated Ag RDT appeared to be suitable for fast identification of acute infections associated with high viral loads. Market access of SARS-CoV-2 Ag RDT should be based on minimal requirements for sensitivity and specificity.

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“…There is convincing evidence that infectivity of SARS-CoV-2 correlates directly with high viral loads in respiratory specimens of acutely infected persons [11][12][13].…”

Section: Discussionmentioning

confidence: 99%

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

et al. 2021

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“…Previous studies have revealed that a minimal RNA genome copy number of 10 6 genome copies per mL of specimen represents the amount of infectious virus particles required for successful virus propagation in cell culture [9,[11][12][13][14]. To correlate the pools to potential infectivity in a specimen, we propagated the pools with ≥ 10 6 genome copies per mL, corresponding to a quantification cycle (Cq) value < 25, in cell culture.…”

Section: Evaluation Panelmentioning

confidence: 99%

Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

et al. 2021

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Introduction The detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach. Aim We aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests. Methods While for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories. Results Our results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%. Conclusions Sensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.

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“…cDNA synthesis was performed according to the SuperScript IV Reverse Transcriptase protocol (Thermo Fisher Scientific, Waltham, MA, United States) with random hexamers (65 • C for 5 min and 23 • C for 10 min), followed by incubation at 55 • C for 10 min and inactivation at 80 • C for 10 min. Relative genome concentration of all strains was determined by specific quantitative real-time PCR (Michel et al, 2021). The performance of the AmpliCoV sequencing was further tested on clinical specimens (n = 36) from patients with a clinical and laboratory diagnosis of COVID-19.…”

Section: Evaluation Of Amplicov Sequencing With Cell Culture-propagated Virus Strains and Human Clinical Specimensmentioning

confidence: 99%

AmpliCoV: Rapid Whole-Genome Sequencing Using Multiplex PCR Amplification and Real-Time Oxford Nanopore MinION Sequencing Enables Rapid Variant Identification of SARS-CoV-2

et al. 2021

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Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants.

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Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Cited by 46 publications

References 19 publications

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

AmpliCoV: Rapid Whole-Genome Sequencing Using Multiplex PCR Amplification and Real-Time Oxford Nanopore MinION Sequencing Enables Rapid Variant Identification of SARS-CoV-2

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