TargetPlex FFPE-Direct DNA Library Preparation Kit for SiRe NGS panel: an international performance evaluation study.

AIM: Next generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow. METHODS: The TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols. RESULTS: Overall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants. CONCLUSION: The TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.

Quantitative and Ultrasensitive In-situ Immunoassay Technology for SARS-CoV-2 Detection in Saliva.

The COVID-19 pandemic has become an immense global health crisis. However, the lack of efficient and sensitive on-site testing methods limits early detection for timely isolation and intervention. Here, we present a Quantitative and Ultrasensitive in-situ Immunoassay Technology for SARS-CoV-2 detection in saliva (QUIT SARS-CoV-2). Our nanoporous membrane resonator generates a rapid oscillating flow to purify and concentrate SARS-CoV-2 virus in saliva by 40 folds for in-situ detection of viral antigens based on chemiluminescent immunoassay within 20 min. This method achieved a detection sensitivity below 10 0 copies/mL viral load, comparable to the bench-top PCR equipment. The portable QUIT SARS-CoV-2 system, allowing rapid and accurate on-site viral screen with high-throughput sample pooling strategy, can be performed at the primary care settings and substantially improve the detection and prevention of COVID-19.

Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens.

BACKGROUND: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. METHODS: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). RESULTS: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. CONCLUSIONS: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Performance evaluation of the VERSANT HCV RNA qualitative assay by using transcription-mediated amplification.

A preclinical evaluation of a qualitative assay for the detection of hepatitis C virus (HCV) RNA by transcription-mediated amplification (TMA) was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards and the U.S. Food and Drug Administration. Our results showed that this assay, HCV TMA, detected 95% of samples with HCV RNA concentrations of 5.3 IU/ml and 29 copies/ml. HCV TMA showed an overall specificity of 99.6% and was highly reproducible, detecting 99.3% of samples with HCV RNA concentrations of 50 copies/ml across seven different lots of reagents. Experiments with clinical samples showed that HCV TMA detected all HCV genotypes with similar efficiencies, detecting > or = 95% of samples at 50 HCV RNA copies/ml from patients infected with HCV genotypes 1a, 2b, 3a, 4a, 5a, and 6a. In experiments with RNA transcripts, HCV TMA detected > or = 96.6% of transcripts derived from HCV genotypes 1a, 1b, 2a, 2c, 3a, 4a, 5a, and 6a at 50 HCV RNA copies/ml. Detection of transcripts derived from HCV genotype 2b was slightly lower (88.4%) at 50 copies/ml but was 97.0% at 75 copies/ml. In addition, HCV TMA exhibited robust performance in detecting HCV RNA in samples subjected to various conditions commonly encountered in a clinical laboratory, including long-term storage, multiple freeze-thaw cycles, different collection tubes, and the presence of endogenous substances, commonly prescribed drugs, or other microorganisms and viruses. With its high sensitivity, specificity, reproducibility, and equivalent genotype reactivity, HCV TMA may provide an attractive alternative for routine qualitative HCV RNA testing in clinical laboratories.