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.
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 >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 >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.As global population estimates reach 170 million infected with the hepatitis C virus (HCV) (23), there has never been a more pressing need for sensitive, precise tests for active infections. Although enzyme immunoassays (EIA) followed by confirmatory immunoblot assays have been traditionally used for screening and testing of blood, neither assay can differentiate between active and resolved infection. Qualitative and quantitative HCV RNA testing as well as HCV antigen detection methods can identify active infection, but with quantitative tests usually being 1 to 2 logs less sensitive than qualitative tests and with the limited availability of antigen methods, qualitative HCV RNA testing is the method of choice for confirming active infection and assessing viral clearance in response to therapy (8).Qualitative HCV RNA assays currently used are based on PCR technology and include the AMPLICOR HCV 2.0,
AimNext 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.MethodsThe 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.ResultsOverall, 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.ConclusionThe 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.
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.
Translational and clinical cancer research require rapid approaches that are compatible with small input amounts of challenging research samples such as FFPE tissue. By overcoming known barriers of multiplex PCR, the Ion AmpliSeq™ technology introduces a groundbreaking workflow enabling the rapid sequencing of hundreds of known mutations with low allele frequency with detection down to 5%. Utilizing low input DNA, this single-tube, single day workflow is as simple as setting up a PCR reaction. The Ion AmpliSeq™ Cancer Panel is a fixed-content panel that matches the specific needs of clinical and translational researchers. Rapid and parallel assessment of 739 key somatic and germline mutations in 46 gene targets is enabled. Simple workflow allows library preparation in just 3.5 hours. Comparisons will be presented on results from tumor/normal pairs on FFPE DNA samples from various cancers samples including lung, thyroid and colon. The Ion AmpliSeq™ Custom Assays allows cancer researchers the ability to select targets of choice to accommodate between 10 kb and 1 Mb of cumulative human genome coverage. Using the web-based Torrent Assay Designer, researchers can quickly submit gene names and/or gene coordinates up to 768 amplicon per pool and multiple pools can be combined to run on a single Ion chip allowing up 12,288 amplicons to be assayed at once. Results will be presented on a 768 amplicon panel being used to test several tumor vs normal paired samples. Method: Genomic DNA was extracted from FFPE samples using the PureLink® Genomic DNA Mini Kit. Samples were processed according to the Ion AmpliSeq™ Library Preparation User Guide. The extracted DNA was quantitated with the Qubit® Fluorometer. Ten ng of DNA was amplified via PCR using a master mix of 190 or 768 cancer primer pairs. The amplicons were then purified and phosphorylated. Template preparation automated on the Ion OneTouch™ System. After template preparation the amplicon library from each sample was loaded onto an Ion 314™ chip and sequenced in 90 minute on the Ion PGM™ sequencer. The Torrent Suite Software was used for variant calling via the AmpliSeq Cancer Variant plug-in. Results & Conclusions: We detected 100% of the known somatic mutations and detected no false-positives. The Ion AmpliSeq™ technology has shown itself to be the fastest and simplest approach to generating amplicon libraries of targeted regions of interest. The Ion AmpliSeq™ Cancer Panel is capable of detection 739 known Cosmic mutations across 46 key cancer genes in a single day with high sensitivity and high accuracy. The Ion AmpliSeq™ Custom assays enable quick custom assay design turn-around and accurate data generation for fast project completion. Details on further fixed-content cancer panels will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3182. doi:1538-7445.AM2012-3182
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.