RESUMO
Currently, testing for coronavirus is performed with time and personnel consuming PCR assays. The aim of this study was to evaluate the sensitivity, specificity and capacity of a fully automated, random access high-throughput real-time PCR-based diagnostic platform for the detection of SARS-CoV-2. The NeuMoDx N96 system displayed an equal or better detection rate for SARS-CoV-2 compared with the LightCycler 480II system and showed a specificity of 100%. The median PCR run time for all 28 PCR runs was 91 (IQR 84-97) minutes. The capacity of the NeuMoDx N96 could easily surpass the capacity of most currently used molecular test systems and significantly reduce the turn-around time.
Assuntos
Betacoronavirus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , SARS-CoV-2 , Sensibilidade e Especificidade , Fatores de TempoRESUMO
The analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood can serve as a minimally invasive alternative to tumor tissue biopsies in cases with impaired access to tissue. Its clinical utility has been well demonstrated for EGFR T790M testing in lung cancer patients suffering progress after tyrosine kinase inhibitor treatment. At present, highly sensitive unique molecular identifiers (UMI)-based NGS for liquid biopsy testing is less established compared to single gene assays. However, the critical bottleneck are sufficient cfDNA yields, which are essentially required to obtain meaningful test results. We compared four different cfDNA extraction methods (Qiagen, Promega, Thermo and Stratec) using the same plasma samples in order to evaluate their suitability for further NGS analysis. We managed to draw 60 ml blood from 12 patients each and equally collected 30ml in PAXgene and EDTA tubes at the same time point, sufficient for total of 96 cfDNA extractions. CfDNA concentrations and total amounts were highest for Qiagen and Promega protocols, showing the best read length profiles after sequencing. Known oncogenic driver mutations were identified in 9 out of 12 patients with at least one of the cfDNA extraction methods, again favoring the extraction protocols from Qiagen and Promega. We also uncovered putative sequencing artefacts including known driver genes pointing to a careful consideration for the limit of detection of this methodology. Our study shows that pre-analytical optimization is necessary to achieve the maximum sensitivity of UMI-based sequencing but also highlights the low abundance of tumor-derived cfDNA in lung cancer samples.
