scholarly journals Coupling multiplex pre-amplification and droplet digital PCR for longitudinal monitoring ofESR1andPIK3CAmutations from plasma cell-free DNA

2019 ◽  
Author(s):  
Huilan Yao ◽  
Grant Wu ◽  
Subhasree Das ◽  
Crystal MacKenzie ◽  
Hua Gao ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Hot Spot ◽  
Metastatic Breast ◽  
High Sensitivity ◽  
Cost Effective ◽  
Digital Pcr ◽  
Cost Effective Method ◽  
High Prevalence ◽  
High Concordance ◽  
Hotspot Mutations

AbstractHere we report on the development of a sensitive and cost-effective method to longitudinally trackESR1andPIK3CAmutations from cfDNA in patients with metastatic breast cancer (MBC) using a streamlined and de-centralized workflow. Hotspot mutations inESR1have been shown to cause resistance to aromatase inhibitor–based and anti-estrogenic therapies, whilePIK3CAmutations have high prevalence in MBC. As a result, their utility as circulating biomarkers to predict or monitor response in the clinical development of investigational compounds has been the focus of many studies. Six regions inESR1andPIK3CAgenes containing 20 hotspot mutations were pre-amplified, followed by optimized singleplex ddPCR assays to detect allele frequencies of individual mutations. Without pre-amplification, the limit of detection (LOD) and limit of linearity (LOL) of individual ddPCR assays were at 0.05-0.1% and 0.25% level, respectively. With pre-amplification, the LOD and LOL were slightly elevated at 0.1-0.25% and 0.25-0.5% levels, respectively. High concordance was achieved to the BEAMing assay (Sysmex Inostics) for mutation positive assays (r=0.98, P<0.0001). In conclusion, coupling pre-amplification and ddPCR assays allowed us for the detection of up to 20 hot spot mutations inESR1andPIK3CAwith high sensitivity and reproducibility.

scholarly journals Fabrication and Characterization of Humidity Sensors Based on Graphene Oxide–PEDOT:PSS Composites on a Flexible Substrate

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 148 ◽  
Author(s):  
Francisco J. Romero ◽  
Almudena Rivadeneyra ◽  
Markus Becherer ◽  
Diego P. Morales ◽  
Noel Rodríguez
Keyword(s):  
Graphene Oxide ◽  
Large Scale ◽  
Flexible Substrate ◽  
High Sensitivity ◽  
Cost Effective ◽  
Humidity Sensors ◽  
Good Thermal Stability ◽  
Cost Effective Method ◽  
And Performance ◽  
Stability Time

In this paper, we present a simple, fast, and cost-effective method for the large-scale fabrication of high-sensitivity humidity sensors on flexible substrates. These sensors consist of a micro screen-printed capacitive structure upon which a sensitive layer is deposited. We studied two different structures and three different sensing materials by modifying the concentration of poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT:PSS) in a graphene oxide (GO) solution. The results show that the aggregation of the PEDOT:PSS to the GO can modify its electrical properties, boosting the performance of the capacitive sensors in terms of both resistive losses and sensitivity to relative humidity (RH) changes. Thus, in an area less than 30 mm2, the GO/PEDOT:PSS-based sensors can achieve a sensitivity much higher (1.22 nF/%RH at 1 kHz) than other similar sensors presented in the literature which, together with their good thermal stability, time response, and performance over bending, demonstrates that the manufacturing approach described in this work paves the way for the mass production of flexible humidity sensors in an inexpensive way.

scholarly journals Molecularly Imprinted Nanoparticles Based Sensor for Cocaine Detection

Biosensors ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 22 ◽  
Author(s):  
Roberta D’Aurelio ◽  
Iva Chianella ◽  
Jack A. Goode ◽  
Ibtisam E. Tothill
Keyword(s):  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Cost Effective ◽  
Cross Reactivity ◽  
P Value ◽  
Sensing Element ◽  
Molecularly Imprinted ◽  
Cost Effective Method ◽  
Cocaine Detection

The development of a sensor based on molecularly imprinted polymer nanoparticles (nanoMIPs) and electrochemical impedance spectroscopy (EIS) for the detection of trace levels of cocaine is described in this paper. NanoMIPs for cocaine detection, synthesized using a solid phase, were applied as the sensing element. The nanoMIPs were first characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering and found to be ~148.35 ± 24.69 nm in size, using TEM. The nanoMIPs were then covalently attached to gold screen-printed electrodes and a cocaine direct binding assay was developed and optimized, using EIS as the sensing principle. EIS was recorded at a potential of 0.12 V over the frequency range from 0.1 Hz to 50 kHz, with a modulation voltage of 10 mV. The nanoMIPs sensor was able to detect cocaine in a linear range between 100 pg mL−1 and 50 ng mL−1 (R2 = 0.984; p-value = 0.00001) and with a limit of detection of 0.24 ng mL−1 (0.70 nM). The sensor showed no cross-reactivity toward morphine and a negligible response toward levamisole after optimizing the sensor surface blocking and assay conditions. The developed sensor has the potential to offer a highly sensitive, portable and cost-effective method for cocaine detection.

scholarly journals STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

2020 ◽  
Author(s):  
Xiaoxian Guo ◽  
Yiqin Wang ◽  
Ruoyu Zhang ◽  
Zhenglong Gu
Keyword(s):  
Large Scale ◽  
Nuclear Dna ◽  
Large Population ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Sequencing Method ◽  
Scale Population ◽  
Mtdna Variants ◽  
Multiplex Sequencing

ABSTRACTHuman mitochondrial genome (mtDNA) variations, such as mtDNA heteroplasmies (the co-existence of mutated and wild-type mtDNA), have received increasing attention in recent years for their clinical relevance to numerous diseases. But large-scale population studies of mtDNA heteroplasmies have been lagging due to the lack of a labor- and cost-effective method. Here, we present a novel human mtDNA sequencing method called STAMP (sequencing by targeted amplification of multiplex probes) for measuring mtDNA heteroplasmies and content in a streamlined workflow. We show that STAMP has high mapping rates to mtDNA, deep coverage of unique reads, and high tolerance to sequencing and PCR errors when applied to human samples. STAMP also has high sensitivity and low false positive rates in identifying artificial mtDNA variants at fractions as low as 0.5% in genomic DNA samples. We further extend STAMP, by including nuclear DNA-targeting probes, to enable assessment of relative mtDNA content in the same assay. The high cost-effectiveness of STAMP, along with the flexibility of using it for measuring various aspects of mtDNA variations, will accelerate the research of mtDNA heteroplasmies and content in large population cohorts, and in the context of human diseases and aging.

scholarly journals A single droplet digital PCR for ESR1 activating mutations detection in plasma

2019 ◽  
Author(s):  
Emmanuelle Jeannot ◽  
Lauren Darrigues ◽  
Marc Michel ◽  
Marc-Henri Stern ◽  
Jean-Yves Pierga ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Metastatic Breast Cancer ◽  
Limit Of Detection ◽  
Growth Factor Receptor ◽  
Metastatic Breast ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Plasma Samples ◽  
Activating Mutations

AbstractBackgroundActivating mutations in the estrogen receptor 1 (ESR1) gene are recurrent mechanisms of acquired resistance to aromatase inhibitors (AI), and may be the target of other selective estrogen receptor down-regulators. To assess the clinical utility of monitoring ESR1 resistant mutations, a droplet digital PCR (ddPCR)-based assay compatible with body fluids is ideal due to its cost-effectiveness and quick turnaround.MethodsWe designed a multiplex ddPCR, which combines a drop-off assay, targeting the clustered hotspot mutations found in exon 8, with another pair of probes interrogating the E380Q mutation in exon 5. We assessed its sensitivity in vitro using synthetic oligonucleotides, harboring E380Q, L536R, Y537C, Y537N, Y537S or D538G mutations. Validation of the assay was performed on plasma samples from a prospective study and compared to next generation sequencing (NGS) data.ResultsThe multiplex ESR1-ddPCR showed a high sensitivity with a limit of detection ranging from 0.07 to 0.19% in mutant allele frequency depending on the mutation tested. The screening of plasma samples from patients with AI-resistant metastatic breast cancer identified ESR1 mutations in 29% of them with perfect concordance (and higher sensitivity) to NGS data obtained in parallel. Additionally, this test identifies patients harboring polyclonal alterations. Furthermore, the monitoring of ctDNA using this technique during treatment follow-up predicts the radiological response to palbociclib-fulvestrant.ConclusionThe multiplex ESR1-ddPCR detects, in a single reaction, the most frequent ESR1 activating mutations and is compatible with plasma samples. This method is thus suitable for real-time ESR1 mutation monitoring in large cohorts of patients.Statement of translational relevanceExons 5 and 8 mutations in ESR1 are recurrent mechanisms of resistance to aromatase inhibitors (AI) in estrogen receptor (ER)-positive metastatic breast cancer and may be targeted by selective ER down-regulators. We implemented a novel droplet digital PCR, which allows for the detection of the most frequent ESR1 mutations in circulating cell-free DNA. In prospectively collected plasma samples, ESR1 mutations were found in 29% of AI-resistant patients, with excellent concordance and higher sensitivity to next generation sequencing. Moreover, circulating ESR1 mutations appear to be reliable markers for ctDNA monitoring in order to predict treatment response. Ultimately, the short turnaround time, high sensitivity and limited cost of the ESR1-ddPCR are compatible with repeated samplings to detect the onset of resistance to AI before the radiological progression. This opens a window of opportunity to develop new clinical strategies for breast cancer hormone therapy, as tested in an ongoing phase 3 trial.List of abbreviationsAIAromatase InhibitorcfDNACell-free DNActDNACirculating tumor DNAddPCRDroplet digital PCRER+ HER2-MBCER+ HER2-negative Metastatic Breast CancerEREstrogen ReceptorER+Estrogen Receptor positiveLOBLimit of blankLODLimit of detectionMAFMutant Allele FrequencyPBMCPeripheral blood mononuclear cellsPDProgressive diseaseSDStandard deviationToPTime of progressionWTWild typeHuman genesESR1: Estrogen Receptor 1HER2: Human Epidermal Growth Factor Receptor 2EGFR: Epithelial Growth Factor ReceptorKRAS: KRAS proto-oncogene, GTPaseBRAF: B-Raf Proto-Oncogene, Serine/Threonine kinase

scholarly journals Diagnostic performance of rapid antigen tests (RATs) for SARS-CoV-2 and their efficacy in monitoring the infectiousness of COVID-19 patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
John G. Routsias ◽  
Maria Mavrouli ◽  
Panagiota Tsoplou ◽  
Kyriaki Dioikitopoulou ◽  
Athanasios Tsakris
Keyword(s):  
Diagnostic Performance ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Cost Effective ◽  
Clinical Samples ◽  
True Positive ◽  
Antigen Tests ◽  
The Cost ◽  
Very High ◽  
Test Result

AbstractThe most widely used test for the diagnosis of SARS-CoV-2 infection is a PCR test. PCR has very high sensitivity and is able to detect very low amounts of RNA. However, many individuals receiving a positive test result in a context of a PCR-based surveillance might be infected with SARS-CoV-2, but they are not contagious at the time of the test. The question arises regards if the cost effective, portable rapid antigen tests (RATs) have a better performance than PCR in identification of infectious individuals. In this direction, we examined the diagnostic performance of RATs from 14 different manufacturers in 400 clinical samples with known rRT-PCR cycles threshold (cT) and 50 control samples. Substantial variability was observed in the limit of detection (LOD) of different RATs (cT = 26.8–34.7). The fluorescence-based RAT exhibited a LOD of cT = 34.7. The use of the most effective RATs leads to true positive rates (sensitivities) of 99.1% and 90.9% for samples with cT ≤ 30 and cT ≤ 33, respectively, percentages that can guarantee a sensitivity high enough to identify contagious patients. RAT testing may also substantially reduce the quarantine period for infected individuals without compromising personal or public safety.

scholarly journals STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

2020 ◽  
Vol 2 (4) ◽  
Author(s):  
Xiaoxian Guo ◽  
Yiqin Wang ◽  
Ruoyu Zhang ◽  
Zhenglong Gu
Keyword(s):  
Large Scale ◽  
Nuclear Dna ◽  
Large Population ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Sequencing Method ◽  
Scale Population ◽  
Mtdna Variants ◽  
Multiplex Sequencing

Abstract Human mitochondrial genome (mtDNA) variations, such as mtDNA heteroplasmies (the co-existence of mutated and wild-type mtDNA), have received increasing attention in recent years for their clinical relevance to numerous diseases. But large-scale population studies of mtDNA heteroplasmies have been lagging due to the lack of a labor- and cost-effective method. Here, we present a novel human mtDNA sequencing method called STAMP (sequencing by targeted amplification of multiplex probes) for measuring mtDNA heteroplasmies and content in a streamlined workflow. We show that STAMP has high-mapping rates to mtDNA, deep coverage of unique reads and high tolerance to sequencing and polymerase chain reaction errors when applied to human samples. STAMP also has high sensitivity and low false positive rates in identifying artificial mtDNA variants at fractions as low as 0.5% in genomic DNA samples. We further extend STAMP, by including nuclear DNA-targeting probes, to enable assessment of relative mtDNA content in the same assay. The high cost-effectiveness of STAMP, along with the flexibility of using it for measuring various aspects of mtDNA variations, will accelerate the research of mtDNA heteroplasmies and content in large population cohorts, and in the context of human diseases and aging.

scholarly journals Droplet-Digital PCR Provides a Rapid, Accurate and Cost-Effective Method for Identification of Biomarker FcγRIIIa-F158V Genotypes

2017 ◽  
Vol 06 (02) ◽  
Author(s):  
Paul Griffith ◽  
David Sun ◽  
Sarah R Tritsch ◽  
Caroline Jochems ◽  
James L Gulley ◽  
...  
Keyword(s):  
Cost Effective ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cost Effective Method

scholarly journals PfHRP2 detection using plasmonic optrodes: performance analysis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Médéric Loyez ◽  
Mathilde Wells ◽  
Stéphanie Hambÿe ◽  
François Hubinon ◽  
Bertrand Blankert ◽  
...  
Keyword(s):  
Optical Fiber ◽  
Limit Of Detection ◽  
Fiber Surface ◽  
Malaria Diagnosis ◽  
High Sensitivity ◽  
Cost Effective ◽  
Label Free ◽  
Multiple Receptors ◽  
Label Free Detection

Abstract Background Early malaria diagnosis and its profiling require the development of new sensing platforms enabling rapid and early analysis of parasites in blood or saliva, aside the widespread rapid diagnostic tests (RDTs). Methods This study shows the performance of a cost-effective optical fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Unclad multimode optical fiber probes are coated with a thin gold film to excite Surface Plasmon Resonance (SPR) yielding high sensitivity to bio-interactions between targets and bioreceptors grafted on the metal surface. Results Their performances are presented in laboratory conditions using PBS spiked with growing concentrations of purified target proteins and within in vitro cultures. Two probe configurations are studied through label-free detection and amplification using secondary antibodies to show the possibility to lower the intrisic limit of detection. Conclusions As malaria hits millions of people worldwide, the improvement and multiplexing of this optical fiber technique can be of great interest, especially for a future purpose of using multiple receptors on the fiber surface or several coated-nanoparticles as amplifiers.

scholarly journals A Simple Colorimetric Molecular Detection of Novel Coronavirus (COVID-19), an Essential Diagnostic Tool for Pandemic Screening

2020 ◽  
Author(s):  
Pazhanimuthu Annamalai ◽  
Madhu Kanta ◽  
Pazhanivel Ramu ◽  
Baskar Ravi ◽  
Kokilavani Veerapandian ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Recent Outbreak ◽  
Pcr Method ◽  
Novel Coronavirus ◽  
Virus Isolates

AbstractThe recent outbreak of the newly emerged novel coronavirus (SARS-CoV-2) presents a big challenge for public health laboratories as virus isolates are not available while there is an increasing evidence that the epidemic is more widespread than initially thought, as well as spreading internationally across borders through travellers does already happen warranting a methodology for the rapid detection of the infection to control SARS-CoV-2. Aim: We intended to develop and deploy a robust and rapid diagnostic methodology using LAMP assay for use in point of care settings to detect SARS-COV-2 infection. Methodology: In the present study, we have developed a validated rapid diagnostic procedure to detect SARS-CoV-2 using LAMP assay, its design relying on isothermal amplification of the nucleic acids of the SARS-CoV-2. Results: The LAMP assay developed detects SARS-CoV-2 infection rapidly with high sensitivity and reliability. The data generated by LAMP assay were comparable and at par with the data generated by real-time PCR method. Conclusion: The present study demonstrates that the LAMP assay developed was a rapid, reliable, sensitive and cost effective method to detect SARS-CoV-2 infection in a point of care as well as in laboratory settings.

Ephedrine Analysis in Real Urine Sample via Solvent Bar Microextraction Technique Coupled with HPLC-UV and Chemometrics

2018 ◽  
Vol 15 (1) ◽  
pp. 24-31 ◽  
Author(s):  
Persia Behbahani ◽  
Mahnaz Qomi ◽  
Nahid Ghasemi ◽  
Kambiz Tahvildari
Keyword(s):  
Urine Sample ◽  
High Performance ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Relative Standard ◽  
Designed Experiment ◽  
Preconcentration Factor ◽  
Beta Adrenergic Agonist ◽  
Solvent Bar Microextraction

Background: Ephedrine, an alpha/beta-adrenergic agonist, is one of the most common doping agents not only among athletes but also the ordinary people, therefore its detection at low trace levels with a sensitive and cost effective method has become a priority to investigate many analytical methods. Objective: In this work, solvent bar microextraction followed by high-performance liquid chromatography (HPLC-UV) was used for extraction and determination of ephedrine at low trace levels from urine samples at optimum condition. Methods: In this study, a designed experiment was carried out using solvent bar microextraction technique, which has been proved to be a green method. This method requires three phases consisting of a donor phase with an alkaline pH, an acceptor phase with an acidic pH, and organic solvent to impregnate the pores of the hollow fiber. The obtained results were used for estimating the optimum ranges for each parameter, analyzing the effect of different parameters, simultaneously. Results: Under optimized circumstances, the preconcentration factor was 129. The calibration curves represented good linearity for urine sample with coefficient estimations higher than 0.9991. The limit of detection and quantitation for ephedrine were 16.7 µg L-1 and 50 µg L-1, respectively. The relative standard deviations of analysis were 3.5% within a day (n=3) and 4.1% between days (n=9). Conclusion: According to the results and previous studies, it can be concluded that the preconcentration factor for ephedrine was the best result ever reported considering selectivity and cost-effectiveness.

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