scholarly journals Real-time colorimetric LAMP methodology for quantitative nucleic acids detection at the point-of-care

2020 ◽  
Author(s):  
George Papadakis ◽  
Alexandros K. Pantazis ◽  
Nikolaos Fikas ◽  
Stella Chatziioannidou ◽  
Kleita Michaelidou ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Real Time ◽  
Dynamic Range ◽  
Point Of Care ◽  
Cost Effective ◽  
Detection Capability ◽  
Ability To Work ◽  
Rna Detection ◽  
Fast Prototyping ◽  
Nucleic Acids Detection

AbstractMost methods applied to nucleic acids’ detection at the point-of-care require either expensive and mostly bench-top instruments or simpler inexpensive systems providing qualitative results. Truly decentralized approaches to reliable, quantitative and affordable diagnostics are still missing. Here, we report the development of real-time quantitative colorimetric LAMP based on a portable and cost-effective device, the use of which requires minimal training. Main advantages of the method are the rapid analysis time (<30min); quantification over a large dynamic range (9 log units); ability to work with crude samples (saliva, tissue); demonstrated low detection limit (1-10 copies); smartphone-operation and fast prototyping (3D-printing). The system’s broad detection capability is demonstrated during infectious diseases-testing for COVID-19 and pharmacogenetics for BRAF V600E mutation testing. Validation studies showed 97.4% and 100% agreement with qRT-PCR for SARS-CoV-2 RNA detection extracted from positive and negative patients’ samples (89), respectively; and 100% agreement with ddPCR and Sanger sequencing for BRAF V600E mutation detection from 12 clinical biopsy samples. The new methodology provides a needed solution for affordable healthcare at the point-of-care, with emphasis on global diagnostics.

Comparison of digital PCR with real-time PCR calibrated to the International Scale for BCR-ABL monitoring.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e18545-e18545
Author(s):  
Parth Shah ◽  
Furha Iram Cossor ◽  
Shiva Murarka ◽  
Hemangi Dixit
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Residual Disease ◽  
Pearson Correlation ◽  
Cost Effective ◽  
Digital Pcr ◽  
Absolute Quantitation ◽  
International Scale ◽  
Accurate Quantification

e18545 Background: Monitoring BCR-ABL1 fusion transcripts is the cornerstone of management in chronic myeloid leukemia (CML). Real Time PCR(RTPCR) has been the tool of choice for its sensitivity and dynamic range. The International Scale was recently introduced to allow for standardization across laboratories. Chip based Digital PCR(dPCR) which allows for absolute quantitation may obliviate the need for such standardization by giving absolute results.It has the potential to increase sensitivity for detection of minimal residual disease. This assumes great significance as we move into the era of treatment free remission. Methods: A total of 31 EDTA-blood samples of CML patients with known values ranging from 0.003-0.5 IS were processed via an accredited RTPCR protocol calibrated to the IS scale and a parallel dPCR workflow without any specific calibration . The analysis of digital PCR samples was performed on the Thermofisher Cloud Platform. Results: Both RTPCR and dPCR yielded extremely concordant results. The Pearson correlation between the two methods was r = 0.6043 (95%CI: 0.318 to 0.789; p = 0.0003). The calculated BCR-ABL/ABL ratios were comparable with a median of 0.098 for RT-PCR calibrated to the IS (range 0.003-0.55; n = 26) and 0.11 for dPCR (range 0.003-0.37; n = 29). The mean difference for the ratios between the two methods used for the detection was around 0.08. Conclusions: We demonstrate here the capability of dPCR to provide a parallel result to an IS scale calibrated protocol without any standardization. This approach required no specific calibrators or standards and resulted in extremely cost effective testing. This freedom from routine calibration provides for a significantly more robust workflow and greatly increased reliability. Limitations do persist in dPCR on account of limited chip densities which are the main parameter for the Poisson statistics. This has limited the dynamic range on dPCR to a maximum of Log 4 for accurate quantification. However as chip densities and emulsion concentrations increase in these technologies, they are poised to introduce a new era in the quest of accurate quantification.

scholarly journals Manipulation of Magnetic Beads with Thin Film Microelectromagnet Traps

Micromachines ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 607 ◽  
Author(s):  
Vania Silverio ◽  
Miguel Amaral ◽  
João Gaspar ◽  
Susana Cardoso ◽  
Paulo P. Freitas
Keyword(s):  
Real Time ◽  
Magnetic Beads ◽  
Cooling System ◽  
Point Of Care ◽  
Cost Effective ◽  
Local Concentration ◽  
Effective Care ◽  
In Series ◽  
Microfluidic Flows ◽  
System Variables

Integration of point-of-care assays can be facilitated with the use of actuated magnetic beads (MB) to perform testing in less expensive settings to enable the delivery of cost-effective care. In this paper we present six different designs of planar microelectromagnets traps (MEMT) with four external coils in series and one central coil connected for an opposite direction of manipulation of MB in microfluidic flows. The development of a simulation tool facilitated the rapid and efficient optimization of designs by presenting the influence of system variables on real time concentrations of MB. Real time experiments are in good agreement with the simulations and showed that the design enabled synchronous concentration and dispersion of MB on the same MEMT. The yield of local concentration is seen to be highly dependent on coil design. Additional coil turns between the central and external coils (inter-windings) doubled magnetic concentration and repulsion with no significant electrical resistance increase. The assemblage of a copper microchannel closed loop cooling system to the coils successfully eliminated the thermal drift promoted by joule heating generated by applied current.

Reusable Surface Molecular Imprint Biosensors Aided by Naturally Occurring Surface Roughness Indices for Point-of-Care Diagnostics

MRS Advances ◽  
2019 ◽  
Vol 4 (22) ◽  
pp. 1299-1308 ◽  
Author(s):  
Yehoshua Auerbach ◽  
Rebecca Isseroff ◽  
Nicholas Clayton ◽  
Miguel Hulyalkar ◽  
Andrew Todt ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Imprinting ◽  
Point Of Care ◽  
High Sensitivity ◽  
Cost Effective ◽  
Stem Cell Differentiation ◽  
Diverse Range ◽  
Point Of Care Diagnostics ◽  
Molecular Imprint ◽  
Detection Of Biomarkers

ABSTRACTWe have shown that molecular imprinting (MI) technology can be used to produce sensitive, robust, cost-effective biosensing systems with a real-time electrochemical readout that can be utilized for point of care diagnostics. Real time detection of biomarkers is essential when rapid, critical decisions need to be made, such as in situations where public health is threatened. Our biosensor has high sensitivity compared to standard methods like ELISA, and results are obtained within minutes, using inexpensive, accessible potentiometric readout technology. These biosensors utilize surface molecular imprinting of a self-assembling monolayer of hydroxy-terminated alkanethiol chains which form a crystalline ‘lock-and-key’ structure around a target analyte, allowing the sensors to detect and differentiate between bio-macromolecules of similar size and shape with high selectivity and sensitivity. The sensors are extremely versatile and able to detect a diverse range of molecules of varied chemical composition and structure. To fully exploit the sensors’ advantages, especially in remote, economically disadvantaged areas, it is important to quantify their durability and reusability. Biosensor chips were created to test the viability of hemoglobin detection and to evaluate the potential for sensor reusability when washed after detection testing. The successful readsorption of hemoglobin even after washing, accompanied by cyclic voltammetry data indicating the preservation of the SAM, indicate that these biosensors are reusable, significantly augmenting the device’s value. Potential applications include the analysis of complex chemical and biological processes such as stem cell differentiation and on-the-spot detection of diseases such as Zika.

scholarly journals Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Janine Michel ◽  
Markus Neumann ◽  
Eva Krause ◽  
Thomas Rinner ◽  
Therese Muzeniek ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Specimen ◽  
Cost Effective ◽  
Pcr Detection ◽  
Rna Detection ◽  
Diagnostic Pcr ◽  
One Step ◽  
Cost Effective Alternative ◽  
Pcr Assays ◽  
Genomic Regions

Abstract Background The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. Aim Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). Methods Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. Results Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. Conclusion The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.

scholarly journals Towards A Graphene Chip System For Blood Clotting Disease Diagnostics

2020 ◽  
Author(s):  
◽  
Jacob J. Mitchell
Keyword(s):  
Real Time ◽  
Blood Clotting ◽  
Point Of Care ◽  
Cost Effective ◽  
Sampling Point ◽  
Sensor Platform ◽  
Disease Diagnostics ◽  
Highly Sensitive ◽  
Wide Range ◽  
Semiconductor Fabrication

Point of care diagnostics (POCD) allows the rapid, accurate measurement of analytes near to a patient. This enables faster clinical decision making and can lead to earlier diagnosis and better patient monitoring and treatment. However, despite many prospective POCD devices being developed for a wide range of diseases this promised technology is yet to be translated to a clinical setting due to the lack of a cost-effective biosensing platform.This thesis focuses on the development of a highly sensitive, low cost and scalable biosensor platform that combines graphene with semiconductor fabrication tech-niques to create graphene field-effect transistors biosensor. The key challenges of designing and fabricating a graphene-based biosensor are addressed. This work fo-cuses on a specific platform for blood clotting disease diagnostics, but the platform has the capability of being applied to any disease with a detectable biomarker.Multiple sensor designs were tested during this work that maximised sensor ef-ficiency and costs for different applications. The multiplex design enabled different graphene channels on the same chip to be functionalised with unique chemistry. The Inverted MOSFET design was created, which allows for back gated measurements to be performed whilst keeping the graphene channel open for functionalisation. The Shared Source and Matrix design maximises the total number of sensing channels per chip, resulting in the most cost-effective fabrication approach for a graphene-based sensor (decreasing cost per channel from £9.72 to £4.11).The challenge of integrating graphene into a semiconductor fabrication process is also addressed through the development of a novel vacuum transfer method-ology that allows photoresist free transfer. The two main fabrication processes; graphene supplied on the wafer “Pre-Transfer” and graphene transferred after met-allisation “Post-Transfer” were compared in terms of graphene channel resistance and graphene end quality (defect density and photoresist). The Post-Transfer pro-cess higher quality (less damage, residue and doping, confirmed by Raman spec-troscopy).Following sensor fabrication, the next stages of creating a sensor platform involve the passivation and packaging of the sensor chip. Different approaches using dielec-tric deposition approaches are compared for passivation. Molecular Vapour Deposi-tion (MVD) deposited Al2O3 was shown to produce graphene channels with lower damage than unprocessed graphene, and also improves graphene doping bringing the Dirac point of the graphene close to 0 V. The packaging integration of microfluidics is investigated comparing traditional soft lithography approaches and the new 3D printed microfluidic approach. Specific microfluidic packaging for blood separation towards a blood sampling point of care sensor is examined to identify the laminar approach for lower blood cell count, as a method of pre-processing the blood sample before sensing.To test the sensitivity of the Post-Transfer MVD passivated graphene sensor de-veloped in this work, real-time IV measurements were performed to identify throm-bin protein binding in real-time on the graphene surface. The sensor was function-alised using a thrombin specific aptamer solution and real-time IV measurements were performed on the functionalised graphene sensor with a range of biologically relevant protein concentrations. The resulting sensitivity of the graphene sensor was in the 1-100 pg/ml concentration range, producing a resistance change of 0.2% per pg/ml. Specificity was confirmed using a non-thrombin specific aptamer as the neg-ative control. These results indicate that the graphene sensor platform developed in this thesis has the potential as a highly sensitive POCD. The processes developed here can be used to develop graphene sensors for multiple biomarkers in the future.

scholarly journals Handheld real-time PCR device

Lab on a Chip ◽  
2016 ◽  
Vol 16 (3) ◽  
pp. 586-592 ◽  
Author(s):  
Christian D. Ahrberg ◽  
Bojan Robert Ilic ◽  
Andreas Manz ◽  
Pavel Neužil
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Point Of Care ◽  
Measurement Data ◽  
Integrated System ◽  
Fluorescence Measurement ◽  
Detection Capability ◽  
Fully Integrated ◽  
Lcd Display

World's smallest, fully autonomous, handheld real-time PCR was shown in this contribution. The device can quickly process up to four samples at a time with detection capability of a single DNA copy. The fully integrated system includes all required electronics for fluorescence measurement, data viewing (LCD display) and processing, and is ideal for use in small clinics and point-of-care applications.

scholarly journals Scaling diagnostics in times of COVID-19: Colorimetric Loop-mediated Isothermal Amplification (LAMP) assisted by a 3D-printed incubator for cost-effective and scalable detection of SARS-CoV-2

2020 ◽  
Author(s):  
Everardo González-González ◽  
Itzel Montserrat Lara-Mayorga ◽  
Iram Pablo Rodríguez-Sánchez ◽  
Felipe Yee-de León ◽  
Andrés García-Rubio ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Point Of Care ◽  
Three Dimensional ◽  
Cost Effective ◽  
Reaction Scheme ◽  
Ease Of Use ◽  
Isothermal Amplification ◽  
N Protein ◽  
Loop Mediated Isothermal Amplification ◽  
3D Printed

AbstractBy the third week of June 2020, more than 8,500,000 positive cases of COVID-19 and more than 450,000 deaths had been officially reported worldwide. The COVID-19 pandemic arrived in Latin America, India, and Africa—territories in which the mounted infrastructure for diagnosis is greatly underdeveloped. Here, we demonstrate the combined use of a three-dimensional (3D)-printed incubation chamber for commercial Eppendorf PCR tubes, and a colorimetric embodiment of a loop-mediated isothermal amplification (LAMP) reaction scheme for the detection of SARS-CoV-2 nucleic acids. We used this strategy to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and a limited number of RNA extracts from patients, we also demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR. We envision that LAMP may greatly enhance the capabilities for COVID-19 testing in situations where RT-qPCR is not feasible or is unavailable. Moreover, the portability, ease of use, and reproducibility of this strategy make it a reliable alternative for deployment of point-of-care SARS-CoV-2 detection efforts during the pandemics.

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