scholarly journals Ultra-Low-Cost Integrated Silicon-based Transducer for On-Site, Genetic Detection of Pathogens

2020 ◽  
Author(s):  
Estefania Nunez-Bajo ◽  
Michael Kasimatis ◽  
Yasin Cotur ◽  
Tarek Asfour ◽  
Alex Collins ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Low Cost ◽  
High Specificity ◽  
Negative Temperature ◽  
Sample Solution ◽  
Rapid Screening ◽  
Battery Capacity ◽  
Field Samples ◽  
Silicon Based

AbstractRapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention e.g., drug therapy, quarantine, no action etc. when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2 (the pathogen causing COVID-19), and shows no or similar symptoms to other common infections. We report a silicon-based integrated Point-of-Need (PoN) transducer (TriSilix) that can chemically-amplify and detect pathogen-specific sequences of nucleic acids (NA) quantitatively in real-time. Unlike other silicon-based technologies, TriSilix can be produced at wafer-scale in a standard laboratory; we have developed a series of methodologies based on metal-assisted chemical (wet) etching, electroplating, thermal bonding and laser-cutting to enable a cleanroom-free low-cost fabrication that does not require processing in an advanced semiconductor foundry. TriSilix is, therefore, resilient to disruptions in the global supply chain as the devices can be produced anywhere in the world. To create an ultra-low-cost device, the architecture proposed exploits the intrinsic properties of silicon and integrates three modes of operation in a single chip: i) electrical (Joule) heater, ii) temperature sensor (i.e. thermistor) with a negative temperature coefficient that can provide the precise temperature of the sample solution during reaction and iii) electrochemical sensor for detecting target NA. Using TriSilix, the sample solution can be maintained at a single, specific temperature (needed for isothermal amplification of NA such as Recombinase Polymerase Amplification (RPA) or cycled between different temperatures (with a precision of ±1.3°C) for Polymerase Chain Reaction (PCR) while the exact concentration of amplicons is measured quantitatively and in real-time electrochemically. A single 4-inch Si wafer yields 37 TriSilix chips of 10×10×0.65 mm in size and can be produced in 7 hours, costing ~US $0.35 per device. The system is operated digitally, portable and low power – capable of running up to 35 tests with a 4000 mAh battery (a typical battery capacity of a modern smartphone). We were able to quantitatively detect a 563-bp fragment (Insertion Sequence IS900) of the genomic DNA of M. avium subsp. paratuberculosis (extracted from cultured field samples) through PCR in real-time with a Limit-of-Detection of 20 fg, equivalent to a single bacterium, at the 30th cycle. Using TriSilix, we also detected the cDNA from SARS-CoV-2 (1 pg), through PCR, with high specificity against SARS-CoV (2003).

scholarly journals Disposable silicon-based all-in-one micro-qPCR for rapid on-site detection of pathogens

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Estefania Nunez-Bajo ◽  
Alexander Silva Pinto Collins ◽  
Michael Kasimatis ◽  
Yasin Cotur ◽  
Tarek Asfour ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Low Cost ◽  
High Specificity ◽  
Rapid Screening ◽  
Quantitative Detection ◽  
The Novel ◽  
Novel Coronavirus ◽  
Silicon Based ◽  
Specific Sequences

AbstractRapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2, and shows no or similar symptoms to other common infections. Here, we report a disposable silicon-based integrated Point-of-Need transducer (TriSilix) for real-time quantitative detection of pathogen-specific sequences of nucleic acids. TriSilix can be produced at wafer-scale in a standard laboratory (37 chips of 10 × 10 × 0.65 mm in size can be produced in 7 h, costing ~0.35 USD per device). We are able to quantitatively detect a 563 bp fragment of genomic DNA of Mycobacterium avium subspecies paratuberculosis through real-time PCR with a limit-of-detection of 20 fg, equivalent to a single bacterium, at the 35th cycle. Using TriSilix, we also detect the cDNA from SARS-CoV-2 (1 pg) with high specificity against SARS-CoV (2003).

scholarly journals A Real-Time Method for Improving Stability of Monolithic Quartz Crystal Microbalance Operating under Harsh Environmental Conditions

Sensors ◽  
2021 ◽  
Vol 21 (12) ◽  
pp. 4166
Author(s):  
Román Fernández ◽  
María Calero ◽  
Yolanda Jiménez ◽  
Antonio Arnau
Keyword(s):  
Real Time ◽  
Quartz Crystal Microbalance ◽  
Quartz Crystal ◽  
Limit Of Detection ◽  
Quartz Substrate ◽  
Low Cost ◽  
Discrete Wavelet ◽  
Reagent Consumption ◽  
Measurement Cell ◽  
The Stability

Monolithic quartz crystal microbalance (MQCM) has recently emerged as a very promising technology suitable for biosensing applications. These devices consist of an array of miniaturized QCM sensors integrated within the same quartz substrate capable of detecting multiple target analytes simultaneously. Their relevant benefits include high throughput, low cost per sensor unit, low sample/reagent consumption and fast sensing response. Despite the great potential of MQCM, unwanted environmental factors (e.g., temperature, humidity, vibrations, or pressure) and perturbations intrinsic to the sensor setup (e.g., mechanical stress exerted by the measurement cell or electronic noise of the characterization system) can affect sensor stability, masking the signal of interest and degrading the limit of detection (LoD). Here, we present a method based on the discrete wavelet transform (DWT) to improve the stability of the resonance frequency and dissipation signals in real time. The method takes advantage of the similarity among the noise patterns of the resonators integrated in an MQCM device to mitigate disturbing factors that impact on sensor response. Performance of the method is validated by studying the adsorption of proteins (neutravidin and biotinylated albumin) under external controlled factors (temperature and pressure/flow rate) that simulate unwanted disturbances.

scholarly journals Electroanalytical Biosensors for Circulating Tumor DNA Detection—A Brief Review

2021 ◽  
Author(s):  
Zhijia Peng ◽  
Xiaogang Lin ◽  
Weiqi Nian ◽  
Xiaodong Zheng ◽  
Jayne Wu
Keyword(s):  
Real Time ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
High Specificity ◽  
Circulating Tumor Dna ◽  
Minimal Invasive ◽  
Diagnosis And Treatment ◽  
Detection Technology ◽  
Tumor Dna

Early diagnosis and treatment have always been highly desired in the fight against cancer, and detection of circulating tumor DNA (ctDNA) has recently been touted as highly promising for early cancer screening. Consequently, the detection of ctDNA in liquid biopsy gains much attention in the field of tumor diagnosis and treatment, which has also attracted research interest from the industry. However, traditional gene detection technology is difficult to achieve low cost, real-time and portable measurement of ctDNA. Electroanalytical biosensors have many unique advantages such as high sensitivity, high specificity, low cost and good portability. Therefore, this review aims to discuss the latest development of biosensors for minimal-invasive, rapid, and real-time ctDNA detection. Various ctDNA sensors are reviewed with respect to their choices of receptor probes, detection strategies and figures of merit. Aiming at the portable, real-time and non-destructive characteristics of biosensors, we analyze their development in the Internet of Things, point-of-care testing, big data and big health.

scholarly journals RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact

2021 ◽  
Vol 11 ◽  
Author(s):  
Natali Vega-Magaña ◽  
Rocío Sánchez-Sánchez ◽  
Jorge Hernández-Bello ◽  
Alberto Antony Venancio-Landeros ◽  
Marcela Peña-Rodríguez ◽  
...  
Keyword(s):  
Biological Effects ◽  
Low Cost ◽  
Gene Mutations ◽  
High Specificity ◽  
Screening Strategy ◽  
Clinical Impact ◽  
Mexican Population ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Reverse Transcription Pcr

BackgroundSeveral variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.MethodsTargeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.ResultsIn silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.ConclusionThis work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

scholarly journals Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Quantification ofSalmonellaspp.,Escherichia coli, andStaphylococcus aureusin Different Food Matrices: Advantages and Disadvantages

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Amanda Teixeira Sampaio Lopes ◽  
George Rêgo Albuquerque ◽  
Bianca Mendes Maciel
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Limit Of Detection ◽  
Microbiological Quality ◽  
Rapid Screening ◽  
Multiplex Qpcr ◽  
Advantages And Disadvantages ◽  
Simultaneous Quantification ◽  
Polymerase Chain ◽  
Food Matrices

Quantitative real-time polymerase chain reactions (qPCRs) of the most prevalent bacteria causing foodborne diseases worldwide, such asSalmonellaspp.,Escherichia coli, andStaphylococcus aureus,can be an important tool for quantitative microbial risk assessment, which requires numerical data to determine the level of contamination at a specific stage of food production. However, most of qPCR assays described in the literature for these pathogens are qualitative; their objective is pathogen detection and not pathogen quantification. Thus, the aim of our work was to develop a qPCR for the simultaneous quantification ofSalmonellaspp.,E. coli, andS. aureusand to propose its use in the analysis of foods, as a tool for microbiological quality monitoring. For this, a multiplex qPCR was standardized for the simultaneous quantification of specific fragments of target genes (ssf,phoA, andnuc) corresponding to each one of the mentioned bacteria. The limit of detection of the technique was 13, 10, and 12 gene copies forssf,phoA, andnuc, respectively; standard curves showed R2> 0.99, with efficiencies ranging from 99 to 110%, and inter- and intraexperiment reproducibility presented a low coefficient of variation in all trials. This methodology was applied in different food matrices (milk, ground beef, and oyster meat), and the results were compared with official microbiological culture methodology and with ready-to-use test. Advantages and disadvantages of each methodology used in this study are pointed out. We suggest that this multiplex qPCR can be used as a rapid screening technique for the analysis of food microbiological quality.

A Simple Paper-Based Lab-on-a-Chip for the Detection of a Highly Pathogenic Strain of Porcine Reproductive and Respiratory Syndrome Virus

2014 ◽  
Vol 67 (10) ◽  
pp. 1434 ◽  
Author(s):  
Piyasak Chaumpluk ◽  
Annop Suriyasomboon
Keyword(s):  
Signal Detection ◽  
Limit Of Detection ◽  
Low Cost ◽  
Dna Amplification ◽  
High Specificity ◽  
Visual Observation ◽  
Nucleic Acid Amplification ◽  
Pathogenic Strain ◽  
Respiratory Syndrome Virus ◽  
Highly Pathogenic

A paper-based laboratory-on-a-chip assay for the rapid detection of a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV) was developed for the first time. The single-unit chip was simply fabricated using Whatman filter paper and plastic lamination. The chip measured 2.5 × 3.0 cm2 and was divided into two parts, one for nucleic acid amplification and the other for signal detection. The HP-PRRSV assay was performed by specific ORF I Nsp 2 gene amplification via an isothermal reverse transcription loop-mediated DNA amplification platform, whereas the cDNA signal detection was performed by visual observation of colorimetric changes in blue silver nanoplates (AgNPls). Positive results caused non-aggregation of the blue AgNPls on the detection pad, whereas negative results induced colorimetric changes in the AgNPls from blue to colourless on the pad. The assay had a limit of detection of 100 copies of the target Nsp 2 gene and high specificity for other types of infectious viruses. The assay required only one hour to complete. This work demonstrates a simple and rapid assay for viruses using a simple, low-cost, paper-based chip.

scholarly journals Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

2017 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Ery Nourika Alfiraza ◽  
Esti Enjelina ◽  
Deni Pranowo
Keyword(s):  
Real Time ◽  
Annealing Temperature ◽  
Limit Of Detection ◽  
Melting Curve ◽  
High Specificity ◽  
Optimization Method ◽  
Amyl Alcohol ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Temperature Optimization

Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.

scholarly journals Adaptive nanopore sequencing on miniature flow cell detects extensive antimicrobial resistence

2021 ◽  
Author(s):  
Adrian Viehweger ◽  
Mike Marquet ◽  
Martin Hölzer ◽  
Nadine Dietze ◽  
Mathias Pletz ◽  
...  
Keyword(s):  
Real Time ◽  
Hospital Admissions ◽  
Microbial Consortium ◽  
Low Cost ◽  
Flow Cell ◽  
Rapid Screening ◽  
Resistant Bacteria ◽  
Cost Constraints ◽  
Antimicrobial Resistance Genes ◽  
New Type

Rapid screening of hospital admissions to detect asymptomatic carriers of resistant bacteria can prevent pathogen outbreaks. However, the resulting isolates rarely have their genome sequenced due to cost constraints and long turn-around times to get and process the data, limiting their usefulness to the practitioner. Here we use real-time, on-device target enrichment ("adaptive") sequencing on a new type of low-cost nanopore flow cell as a highly multiplexed assay covering 1,147 antimicrobial resistance genes. Using this method, we detected four types of carbapenemase in a single isolate of Raoultella ornithinolytica (NDM, KPC, VIM, OXA). Further investigation revealed extensive horizontal gene transfer within the underlying microbial consortium, increasing the risk of resistance spreading. Real-time sequencing could thus quickly inform how to monitor this case and its surroundings.

scholarly journals Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

Plant Disease ◽  
2020 ◽  
Vol 104 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Joseph D. Carrillo ◽  
Joey S. Mayorquin ◽  
Jason E. Stajich ◽  
Akif Eskalen
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Absolute Quantification ◽  
Coefficient Of Determination ◽  
Fungal Isolation ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Symbiotic Fungi ◽  
Fusarium Dieback ◽  
Platanus Racemosa

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

scholarly journals Development of a Direct Competitive ELISA Kit for Detecting Deoxynivalenol Contamination in Wheat

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 50
Author(s):  
Li Han ◽  
Yue-Tao Li ◽  
Jin-Qing Jiang ◽  
Ren-Feng Li ◽  
Guo-Ying Fan ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
High Performance ◽  
Limit Of Detection ◽  
High Specificity ◽  
Hplc Method ◽  
Rapid Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Detection Range ◽  
Technical Requirements ◽  
Relative Standard

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

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