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 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 Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs

2020 ◽  
Vol 21 (12) ◽  
pp. 4396 ◽  
Author(s):  
Tuna Toptan ◽  
Sebastian Hoehl ◽  
Sandra Westhaus ◽  
Denisa Bojkova ◽  
Annemarie Berger ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
World Health ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
M Gene ◽  
Control And Prevention ◽  
Pcr Assays ◽  
Novel Coronavirus ◽  
Health Organization

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

scholarly journals Chest Radiograph (CXR) Manifestations of the Novel Coronavirus Disease 2019 (Covid-19) — A Mini-Review

2020 ◽  
Vol 16 ◽  
Author(s):  
Wai Yee Chan ◽  
Marlina Tanty Ramli Hamid ◽  
Nadia Fareeda Muhammad Gowdh ◽  
Kartini Rahmat ◽  
Nur Adura Yaakup ◽  
...  
Keyword(s):  
Disease Progression ◽  
Real Time ◽  
Low Cost ◽  
Tertiary Care ◽  
Severe Illness ◽  
Care Hospital ◽  
The Novel ◽  
Rt Pcr ◽  
Chest X Ray ◽  
Novel Coronavirus

Background: Coronavirus disease 2019 (COVID-19) is highly contagious and has claimed more than one million lives, besides causing hardship and disruptions. The Fleischner Society has recommended chest X-ray (CXR) in detecting cases with high risk for disease progression, for triaging suspected patients with moderate-to-severe illness, and to eliminate false negatives in areas with high pre-test probability or limited resources. Although CXR is less sensitive than real-time reverse transcription polymerase chain reaction (RT-PCR) in detecting mild COVID-19, it is nevertheless useful because of equipment portability, low cost and practicality in serial assessments of disease progression among hospitalized patients. Objective: This study aims to review the typical and relatively atypical CXR manifestations of COVID-19 pneumonia in a tertiary care hospital. Methods: The CXRs of 136 COVID-19 patients confirmed through real-time RT-PCR from March to May 2020 were reviewed. Literature search was performed using PubMed. Results: A total of 54 patients had abnormal CXR whilst the others were normal. Typical CXR findings included pulmonary consolidation or ground-glass opacities in a multifocal, bilateral peripheral or lower zone distribution, whereas atypical CXR features comprised cavitation and pleural effusion. Conclusion: Typical findings of COVID-19 infection in chest computed tomography studies can also be seen in CXR. The presence of atypical features is associated with worse disease outcome. Recognition of these features on CXR will improve accuracy and speed of diagnosing COVID-19 patients.

scholarly journals Optimized qRT-PCR approach for the detection of intra- and extra-cellular SARS-CoV-2 RNAs

2020 ◽  
Author(s):  
Tuna Toptan ◽  
Sebastian Hoehl ◽  
Sandra Westhaus ◽  
Denisa Bojkova ◽  
Annemarie Berger ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
World Health ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
M Gene ◽  
Control And Prevention ◽  
Pcr Assays ◽  
Novel Coronavirus ◽  
Health Organization

AbstractThe novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

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 Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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 Dual-Modal Assay Kit for the Qualitative and Quantitative Determination of the Total Water Hardness Using a Permanent Marker Fabricated Microfluidic Paper-Based Analytical Device

Chemosensors ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 97
Author(s):  
Oyejide Damilola Oyewunmi ◽  
Seyed Hamid Safiabadi-Tali ◽  
Sana Jahanshahi-Anbuhi
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Limit Of Detection ◽  
Low Cost ◽  
Water Hardness ◽  
Sample Introduction ◽  
World Health ◽  
Total Hardness ◽  
Quantitative Detection ◽  
Qualitative And Quantitative ◽  
Analytical Device

A dip-and-read microfluidic paper-based analytical device (µPAD) was developed for the qualitative and quantitative detection of the total hardness of water. To create well-defined hydrophobic barriers on filter paper, a regular office printer and a commercially available permanent marker pen were utilized as a quick and simple technique with easily accessible equipment/materials to fabricate µPAD in new or resource-limited laboratories without sophisticated equipment. After a wettability and barrier efficiency analysis on the permanent marker colors, the blue and green ink markers exhibited favorable hydrophobic properties and were utilized in the fabrication of the developed test devices. The device had five reaction and detection zones modeled after the classification given by the World Health Organization (WHO), so qualitatively it determined whether the water was ‘soft’, ‘moderately hard’, ‘hard’, or ‘very hard’ by changing color from blue to pink in about 3 min. The device was also used to introduce an alternative colorimetric reaction for quantitative analysis of the water hardness without the need for ethylenediaminetetraacetic acid (EDTA) and without compromising the simplicity and low cost of the device. The developed µPAD showed a calculated limit of detection (LOD) of 0.02 mM, which is at least 80% less than those of commercially available test strips and other reported µPADs, and the results of the real-world samples were consistent with those of the standard titration (with EDTA). In addition, the device exhibited stability for 2 months at room and frigid condition (4 °C) and at varying harsh temperatures from 25 to 100 °C. The results demonstrate that the developed paper-based device can be used for rapid, on-site analysis of water with no interferences and no need for a pipette for sample introduction during testing.

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.

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