scholarly journals Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Patrick Hardinge ◽  
Divesh K. Baxani ◽  
Thomas McCloy ◽  
James A. H. Murray ◽  
Oliver K. Castell
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Soft Matter ◽  
Low Cost ◽  
Diagnostic Technique ◽  
Dna Amplification ◽  
Droplet Microfluidics ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Artificial Cells

AbstractMicrofluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

A Low-Cost and High-Throughput FPGA Implementation of the Retinex Algorithm for Real-Time Video Enhancement

2020 ◽  
Vol 28 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Jin Woo Park ◽  
Hyokeun Lee ◽  
Boyeal Kim ◽  
Dong-Goo Kang ◽  
Seung Oh Jin ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Low Cost ◽  
Fpga Implementation ◽  
Video Enhancement ◽  
Retinex Algorithm

scholarly journals A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

Sensors ◽  
2015 ◽  
Vol 15 (9) ◽  
pp. 23418-23430 ◽  
Author(s):  
Pascal Craw ◽  
Ruth Mackay ◽  
Angel Naveenathayalan ◽  
Chris Hudson ◽  
Manoharanehru Branavan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Low Cost ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification

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 Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

2009 ◽  
Vol 107 (5) ◽  
pp. 1433-1439 ◽  
Author(s):  
K.L. Leung ◽  
C.W. Yip ◽  
W.F. Cheung ◽  
A.C.T. Lo ◽  
W.M. Ko ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Method

scholarly journals System Architecture for IIoT-Based POC Molecular Diagnostic Device

2021 ◽  
Vol 6 (1) ◽  
pp. 60
Author(s):  
Byeong-Heon Kil ◽  
Ji-Seong Park ◽  
Chan-Young Park ◽  
Yu-Seop Kim ◽  
Jong-Dae Kim
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Diagnostic System ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
System Structure ◽  
World Health ◽  
Molecular Diagnostic ◽  
Target System ◽  
Open Platform

In this paper, we investigate an efficient structure for a point-of-care (POC) molecular diagnostic system based on the industrial Internet of things (IIoT). The target system can perform automated molecular diagnosis including DNA extraction, PCR amplification, and fluorescence detection. Samples and reagents are placed in a multi-room cartridge and loaded into the system. A rotating motor and a syringe motor control the cartridge to extract DNA from the sample. The extracted DNA is transferred to a polymerase chain reaction (PCR) chamber for DNA amplification and detection. The proposed system provides multiplexing of up to four colors. For POC molecular diagnostics, the World Health Organization demands features such as low volume, low cost, fast results, and a user-friendly interface. In this paper, we propose a system structure that can satisfy these requirements by using a PCR chip and open platform. A distributed structure is adopted for the convenience of maintenance, and a web-based GUI is adopted for the user’s convenience. We also investigated communication problems that may occur between system components. Using the proposed structure, the user can conveniently control from standard computing devices including a smartphone.

scholarly journals Multiple Compact Camera Fluorescence Detector for Real-Time PCR Devices

Sensors ◽  
2021 ◽  
Vol 21 (21) ◽  
pp. 7013
Author(s):  
Seul-Bit-Na Koo ◽  
Hyeon-Gyu Chi ◽  
Jong-Dae Kim ◽  
Yu-Seop Kim ◽  
Ji-Sung Park ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Performance ◽  
Signal To Noise Ratio ◽  
Low Cost ◽  
Optical Element ◽  
Dna Amplification ◽  
Biological Research ◽  
Fluorescence Detector ◽  
Fluorescence Excitation

The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.

scholarly journals Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

2020 ◽  
Author(s):  
Pierre Garneret ◽  
Etienne Coz ◽  
Elian Martin ◽  
Jean-Claude Manuguerra ◽  
Elodie Brient-Litzler ◽  
...  
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Rna Extraction ◽  
Dna Amplification ◽  
Developed Countries ◽  
Temperature Cycling ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Molecular Testing

In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called COVIDISC to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour opens up a new efficient approach to manage the pandemics.

scholarly journals Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples

2006 ◽  
Vol 52 (4) ◽  
pp. 624-633 ◽  
Author(s):  
Jin Li ◽  
Fengfei Wang ◽  
Harvey Mamon ◽  
Matthew H Kulke ◽  
Lyndsay Harris ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genetic Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Primers ◽  
Medical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Correct Identification

Abstract Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. Methods: We modified one of the 2 PCR primers by use of an oligonucleotide “tail” fluorescently labeled at the 5′ end. An oligonucleotide complementary to this tail, carrying a 3′ quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r2 &gt;0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

scholarly journals Performance assessment of the Allplex™ STI Essential real-time PCR assay for the diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infections in genital and extra-genital sites

2019 ◽  
Vol 43 (4) ◽  
pp. 191-200
Author(s):  
Sylvain Robinet ◽  
François Parisot
Keyword(s):  
Detection Limit ◽  
Chlamydia Trachomatis ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Evaluation Agency ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Standard Curve ◽  
Commensal Flora

Abstract Background Commercial kits performing Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) nucleic acid amplification tests (NAATs) for genital samples are recommended in association with culture, but the majority of real-time polymerase chain reaction (PCR) methods have not received regulatory approval for diagnostics in extra-genital sites. Since 2017, only the Hologic® Aptima Combo2 assay has an in vitro diagnostic (IVD) certification from the European Medicine Evaluation Agency. Methods We assessed the Allplex™ STI-Essential Assay (EA) for the diagnosis of NG and CT in both genital and extra-genital sites. The performance of the extraction step was studied by means of a standard curve between the concentration of expected cultivable gonococci and the cycle threshold (Ct). Three later-generation NAATs were used as comparators, particularly to assess the specificity (Sp). Results A relation between the gonococcal concentration, expressed as colony-forming unit (CFU) per milliliter logarithm, and the Ct was shown to be linear irrespective of the matrices (95% confidence interval [CI]). The detection limit was 10 CFU/mL, contrasting with the relatively poor sensitivity of culture due to inhibitory effects such as pH and the overgrowth of the commensal flora. NG molecular diagnostic is complex and the method comparisons showed some discrepancies when Ct was above 34. We decided to include interpretative comments on our reports on the basis of the Ct result. For CT, comparisons displayed a satisfactory agreement, and the detection limit was 50 copies/mL. Conclusions The Seegene Allplex™ STI-EA showed acceptable performance characteristics for the detection of genital and extra-genital NG and CT.

scholarly journals Development of a Low-Cost, Wireless Smart Thermostat for Isothermal DNA Amplification in Lab-On-A-Chip Devices

Micromachines ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 437
Author(s):  
Pardy ◽  
Sink ◽  
Koel ◽  
Rang
Keyword(s):  
Dna Analysis ◽  
Low Cost ◽  
Lab On A Chip ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Battery Life ◽  
Worst Case ◽  
3D Printed ◽  
Living Organisms ◽  
Smart Thermostat

Nucleic acid amplification tests (NAAT) are widely used for the detection of living organisms, recently applied in Lab-on-a-Chip (LoC) devices to make portable DNA analysis platforms. While portable LoC-NAAT can provide definitive test results on the spot, it requires specialized temperature control equipment. This work focuses on delivering a generalized low-cost, wireless smart thermostat for isothermal NAAT protocols in 2 cm × 3 cm LoC cartridges. We report on the design, prototyping, and evaluation results of our smart thermostat. The thermostat was evaluated by experimental and simulated thermal analysis using 3D printed LoC cartridges, in order to verify its applicability to various isothermal NAAT protocols. Furthermore, it was tested at the boundaries of its operating ambient temperature range as well as its battery life was evaluated. The prototype thermostat was proven functional in 20–30 °C ambient range, capable of maintaining the required reaction temperature of 12 isothermal NAAT protocols with 0.7 °C steady-state error in the worst case.

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