Performance study of an amplification-based NGS test on clinical FFPE specimens in China’s first multi-center study.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e13112-e13112
Author(s):  
Feng Ye ◽  
Xiaoyan Zhou ◽  
Huaiyin Shi ◽  
Haijing Liu ◽  
Li Liang ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Illumina Miseq ◽  
Analytical Sensitivity ◽  
Performance Study ◽  
Ion Torrent Pgm ◽  
Whole Process ◽  
Ffpe Samples ◽  
Ffpe Tissues ◽  
Multi Center Study ◽  
Hotspot Mutation

e13112 Background: Next Generation Sequencing (NGS) assays provide a comprehensive view of clinically actionable variations in patients. The increasing application of NGS holds new promise towards accurate diagnosis, personalized treatment and precision medicine. Formalin-fixed Paraffin-embedded (FFPE) treatment remains to be the most popular format of tissue preservation. To further standardize the whole process of the application of NGS assays on FFPE tissues, the Chinese Medical Association Pathology Division organized a multi-center performance study of a NGS assay on more than 1,000 clinical FFPE specimens by inviting top pathology departments in the country. Methods: OncoAim Tumor Mutation and PharmGx Detection Kit (Singlera Genomics, Shanghai) was used for this study. Reference samples with known mutations and allele frequencies together with 1045 clinical FFPE specimens from 11 participating hospitals were tested. Among them, 615 Colorectal Cancer (CRC) and 430 Non-Small Cell Lung Cancer (NSCLC) FFPE samples were processed and sequenced on Illumina Miseq platform and Thermo ion torrent PGM platform, respectively. Results: The results on reference materials show 100% (Confidence interval = 100%) analytical sensitivity for mutations with MAF ≥ 10% when median coverage is ≥ 500X.Positive predictive value of > 99% was observed for mutations with MAF at 5% (data not shown). The median depth for CRC and NSCLC samples were 977Xand 828X, respectively. Among all CRC samples, 564 (91.7%) samples were found containing at least 1 clinical hotspot alteration. The top mutated genes were TP53, KRAS, APC, PIK3CA, SMAD4, BRAF, FBXW7, NRAS, PTEN, and ERBB2. For all the NSCLC samples, 304 (70.69%) were detected containing at least 1 clinical hotspot mutation. The top 10 mutated genes were EGFR, TP53, KRAS, PIK3CA PTEN, VHL, ERBB2, SMAD4, NFE2L2 and CTNNB1. Conclusions: In this study, we showed that the amplification-based NGS assay can achieve very high sensitivity and specificity for samples with median depth over 500X. The samples containing at least 1 clinical hotspot mutation were about 83% on average, suggesting NGS could be of great use for further characterization of the tissues.

Modelling of the whole process of a university campus CHP power plant and dynamic performance study

2016 ◽  
Vol 13 (1) ◽  
pp. 53-63 ◽  
Author(s):  
Yue Wang ◽  
Akmaral Bermukhambetova ◽  
Ji-Hong Wang ◽  
Mark Donner ◽  
Jun-Fu Lv ◽  
...  
Keyword(s):  
Power Plant ◽  
Dynamic Performance ◽  
University Campus ◽  
Performance Study ◽  
Whole Process

scholarly journals High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Y. Cheng ◽  
Lauren E. Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael T. Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

AbstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

High-sensitivity troponin assays: analytical and clinical aspects

2016 ◽  
Vol 51 (4) ◽  
pp. 315-320
Author(s):  
Magdalena Krintus
Keyword(s):  
Myocardial Infarction ◽  
Clinical Symptoms ◽  
High Sensitivity ◽  
Reference Population ◽  
Clinical Aspects ◽  
Analytical Sensitivity ◽  
Reference Limit ◽  
Highly Sensitive ◽  
Time Required ◽  
Highly Sensitive Troponin

Cardiac troponins are considered the most sensitive and specific biomarkers for the diagnosis of acute coronary syndromes (ACS). According to the Third Universal Definition of Acute Myocardial Infarction, the diagnosis requires a rise / or fall of troponin concentration with at least one value exceeding the 99th percentile upper reference limit (URL) in a reference population with the coexistence of clinical symptoms of ischemia. The introduction of highly sensitive assays has resulted in lower detection limits for the concentration of troponin, allowing for early diagnosis of, as well as the detection of quantifiable concentrations of this biomarker in healthy subjects. According to current guidelines, the use of high-sensitivity tests can shorten the time required to make clinical decisions from the current 3-6 hours to 1-2 hours. The use of highly sensitive troponin assays also carries other potential benefits associated with their predictive value, as well as challenges that include reduced specificity for myocardial infarction, lack of standardization or the presence of biological variability. Given the increasing availability of new, highly sensitive troponin assays we should be aware that their increased analytical sensitivity and precision is accompanied by accurate clinical assessment of the patient, and takes into account other non-cardiac causes of their increased concentrations.

scholarly journals Interpretations of Antibody Responses toSalmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay

1998 ◽  
Vol 5 (4) ◽  
pp. 550-555 ◽  
Author(s):  
Patrick L. McDonough ◽  
Richard H. Jacobson ◽  
John F. Timoney ◽  
Ahmed Mutalib ◽  
David C. Kradel ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
High Sensitivity ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Sensitivity ◽  
Department Of Agriculture ◽  
Trace Back ◽  
Elisa Format ◽  
Commercial Poultry ◽  
Computer Controlled

ABSTRACT Many regulatory and diagnostic programs for the detection ofSalmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a humanS. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemicS. enterica Enteritidis serotype but which also had multiple other salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. entericaEnteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).

Handheld Portable Energy-Dispersive X-Ray Fluorescence Spectrometry (pXRF)

2016 ◽  
pp. 362-381 ◽  
Author(s):  
Elisabeth Holmqvist
Keyword(s):  
Production Systems ◽  
Matrix Effects ◽  
Chemical Characterization ◽  
High Sensitivity ◽  
Limited Range ◽  
Energy Dispersive ◽  
Analytical Sensitivity ◽  
X Ray ◽  
Non Destructive

Handheld portable energy-dispersive X-ray fluorescence (pXRF) spectrometry is used for non-destructive chemical characterization of archaeological ceramics. Portable XRF can provide adequate analytical sensitivity to discriminate geochemically distinct ceramic pastes, and to identify compositional clusters that correlate with data patterns acquired by NAA or other high sensitivity techniques. However, successful non-destructive analysis of unprepared inhomogeneous ceramic samples requires matrix-defined scientific protocols to control matrix effects which reduce the sensitivity and precision of the instrumentation. Quantification of the measured fluorescence intensities into absolute concentration values and detection of light elements is encumbered by the lack of matrix matched calibration and proper vacuum facilities. Nevertheless, semi-quantitative values for a limited range of high Z elements can be generated. Unstandardized results are difficult to validate by others, and decreased analytical resolution of non-destructive surface analysis may disadvantage site-specific sourcing, jeopardize correct group assignments, and lead to under-interpretation of ceramic craft and production systems.

scholarly journals In-Situ Grown Silver Nanoparticles on Nonwoven Fabrics Based on Mussel-Inspired Polydopamine for Highly Sensitive SERS Carbaryl Pesticides Detection

Nanomaterials ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 384 ◽  
Author(s):  
Zhiliang Zhang ◽  
Tiantian Si ◽  
Jun Liu ◽  
Guowei Zhou
Keyword(s):  
Silver Nanoparticles ◽  
Collection Efficiency ◽  
High Sensitivity ◽  
Sers Substrate ◽  
Analytical Sensitivity ◽  
Sers Substrates ◽  
Rapid Sampling ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

The rapid sampling and efficient collection of target molecules from a real-world surface is fairly crucial for surface-enhanced Raman scattering (SERS) to detect trace pesticide residues in the environment and in agriculture fields. In this work, a versatile approach was exploited to fabricate a flexible SERS substrate for highly sensitive detection of carbaryl pesticides, using in-situ grown silver nanoparticles (AgNPs)on non-woven (NW) fabric surfaces based on mussel-inspired polydopamine (PDA) molecules. The obtained NW@PDA@AgNPs fabrics showed extremely sensitive and reproducible SERS signals toward crystal violet (CV) molecules, and the detection limit was as low as 1.0 × 10−12 M. More importantly, these NW@PDA@AgNPs fabrics could be directly utilized as flexible SERS substrates for the rapid extraction and detection of trace carbaryl pesticides from various fruit surfaces through a simple swabbing approach. It was identified that the detection limits of carbaryl residues from apple, orange, and banana surfaces were approximately decreased to 4.02 × 10−12, 6.04 × 10−12, and 5.03 × 10−12 g, respectively, demonstrating high sensitivity and superior reliability. These flexible substrates could not only drastically increase the collection efficiency from multifarious irregular-shaped matrices, but also greatly enhance analytical sensitivity and reliability for carbaryl pesticides. The fabricated flexible and multifunctional SERS substrates would have great potential to trace pesticide residue detection in the environment and bioscience fields.

scholarly journals A Genomic Approach to Develop a New qPCR Test Enabling Detection of the Pyricularia oryzae Lineage Causing Wheat Blast

Plant Disease ◽  
2020 ◽  
Vol 104 (1) ◽  
pp. 60-70 ◽  
Author(s):  
Maud Thierry ◽  
Pierre Gladieux ◽  
Elisabeth Fournier ◽  
Didier Tharreau ◽  
Renaud Ioos
Keyword(s):  
Genomic Analysis ◽  
High Sensitivity ◽  
Fungal Species ◽  
Emerging Diseases ◽  
Pyricularia Oryzae ◽  
Blast Disease ◽  
South American ◽  
Analytical Sensitivity ◽  
Wheat Blast ◽  
Pcr Test

Rapid detection is key to managing emerging diseases because it allows their spread around the world to be monitored and limited. The first major wheat blast epidemics were reported in 1985 in the Brazilian state of Paraná. Following this outbreak, the disease quickly spread to neighboring regions and countries and, in 2016, the first report of wheat blast disease outside South America was released. This Asian outbreak was due to the trade of infected South American seed, demonstrating the importance of detection tests in order to avoid importing contaminated biological material into regions free from the pathogen. Genomic analysis has revealed that one particular lineage within the fungal species Pyricularia oryzae is associated with this disease: the Triticum lineage. A comparison of 81 Pyricularia genomes highlighted polymorphisms specific to the Triticum lineage, and this study developed a real-time PCR test targeting one of these polymorphisms. The test’s performance was then evaluated in order to measure its analytical specificity, analytical sensitivity, and robustness. The C17 quantitative PCR test detected isolates belonging to the Triticum lineage with high sensitivity, down to 13 plasmid copies or 1 pg of genomic DNA per reaction tube. The blast-based approach developed here to study P. oryzae can be transposed to other emerging diseases.

scholarly journals Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

2020 ◽  
Vol 280 ◽  
pp. 113865 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Ion Torrent ◽  
Ion Torrent Pgm
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