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.

Automated diagnosis of Tuberculosis: A review of advancements

2015 ◽  
Vol 1 (2) ◽  
pp. 42-48
Author(s):  
EO Shobowale ◽  
AO Coker ◽  
BA Adegunle
Keyword(s):  
Positive Impact ◽  
Dna Amplification ◽  
High Specificity ◽  
Test Methods ◽  
Ease Of Use ◽  
Nucleic Acid Amplification ◽  
Automated Diagnosis ◽  
Culture Methods ◽  
Control Programs ◽  
Solid Media

Objective: A narrative review of advances in automated diagnostic tests for diagnosis of tuberculosis infections. Methods: Electronic databases were searched for tests on automation in Mycobacterium tuberculosis identication. Studies were selected and evaluated that tested for the performance of new and old methods in automated diagnosis with significant impact on the turn-around time of diagnosis and also positive impact on patient care with respect to outcomes. Results: A total of 40 studies were included. Overall, the gene expert system was found to be superior when applied to respiratory samples as opposed to other body fluids when compared to other test methods. High specificity estimates suggest that  Nucleic Acid Amplification Tests (NAATS) should be the first-line test for rapid diagnosis of meningitis, but that they also need to be combined with the result of other tests in order to rule out disease.  Discussion: Fully automated liquid culture methods overall are superior to mycobacterial culture on solid media, in terms of speed of diagnosis, ease of use and their accuracy with several user friendly systems that can be applied to the Nigerian environment. Conclusion: The DNA amplification tests provide a reliable way of increasing the specificity of diagnosis. Their superior diagnostic capability has been found to hold up in routine clinical practice, and they could confer several advantages on tuberculosis control programs.

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 Reverse Transcription Loop-Mediated Isothermal Amplification for the Detection of Porcine Reproductive and Respiratory Syndrome Virus

2009 ◽  
Vol 21 (3) ◽  
pp. 350-354 ◽  
Author(s):  
Albert Rovira ◽  
Juan Abrahante ◽  
Michael Murtaugh ◽  
Muñoz-Zanzi Claudia
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Restriction Analysis ◽  
Dna Amplification ◽  
Lamp Reaction ◽  
Isothermal Amplification ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Loop Mediated Isothermal Amplification

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine. The objective of the current study is to investigate the feasibility of using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of PRRSV. The RT-LAMP is a recently described DNA amplification technique reported to be simple, inexpensive, fast, and accurate. The RT-LAMP reaction was set up using 2 sets of primers that were designed to detect North American and European strains of PRRSV and performed successfully in a simple heat block. The specificity of the amplified product was demonstrated by restriction analysis. The RT-LAMP was able to detect 5 different PRRSV isolates. However, the limit of detection ranged between 10 2 and 10 4 50% tissue culture infective dose/ml. The RT-LAMP was further evaluated using serum samples from animals of known infection status. The ability of RT-LAMP to detect PRRSV in serum from acutely infected animals was evaluated with 114 serum samples from 18 experimentally inoculated boars. Forty-nine of these samples tested positive by RT-LAMP, while 94 were positive by reverse transcription polymerase chain reaction (RT-PCR). The diagnostic specificity, evaluated with 100 known negative serum samples, was estimated as 99%. The feasibility of RT-LAMP to detect PRRSV was demonstrated in the current study. The RT-LAMP reaction could be performed in just 1 hr with a simple and inexpensive heat block. However, the sensitivity of this technique was significantly lower than that of RT-PCR.

scholarly journals CRISPR/Cas13a powered electrochemical microfluidic biosensor for nucleic acid amplification-free miRNA diagnostics

2019 ◽  
Author(s):  
Richard Bruch ◽  
Julia Baaske ◽  
Claire Chatelle ◽  
Mailin Meirich ◽  
Sibylle Madlener ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Early Stage ◽  
Low Cost ◽  
Clinical Diagnostics ◽  
Nucleic Acid Amplification ◽  
Process Time ◽  
Serum Samples ◽  
Microfluidic Biosensor ◽  
Pcr Method

Non-coding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated to many different diseases, such as cancer, dementia or cardiovascular conditions. The key for an effective treatment is an accurate initial diagnosis at an early stage, improving the patient’s survival chances. Here, we introduce a CRISPR/Cas13a powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a has been realized without any nucleic acid amplification. With a readout time of 9 minutes and an overall process time of less than 4 hours, a limit of detection of 10 pM was achieved, using a measuring volume of less than 0.6 µl. Furthermore, we demonstrate the feasibility of our versatile sensor platform to detect miR-19b in serum samples of children, suffering from brain cancer. The validation of our results with a standard qRT-PCR method shows the ability of our system to be a low-cost and target amplification-free tool for nucleic acid based diagnostics.

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 Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2020 ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Nucleic Acid Amplification ◽  
Nasopharyngeal Swab ◽  
Viral Transport ◽  
Extraction Step ◽  
Highly Sensitive

AbstractThe COVID-19 pandemic has resulted in an urgent global need for rapid, point-of-care diagnostic testing. Existing methods for nucleic acid amplification testing (NAAT) require an RNA extraction step prior to amplification of the viral RNA. This step necessitates the use of a centralized laboratory or complex and costly proprietary cartridges and equipment, and thereby prevents low-cost, scalable, point-of-care testing. We report the development of a highly sensitive and robust, easy-to-implement, SARS-CoV-2 test that utilizes isothermal amplification and can be run directly on viral transport media following a nasopharyngeal swab without the need for prior RNA extraction. Our assay provides visual results in 30 min with 85% sensitivity, 100% specificity, and a limit of detection (LoD) of 2.5 copies/μl, and can be run using a simple heat block.

scholarly journals Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus

Virology ◽  
2013 ◽  
Vol 435 (2) ◽  
pp. 372-384 ◽  
Author(s):  
Baoqing Guo ◽  
Kelly M. Lager ◽  
Jamie N. Henningson ◽  
Laura C. Miller ◽  
Sarah N. Schlink ◽  
...  
Keyword(s):  
United States ◽  
Experimental Infection ◽  
Pathogenic Strain ◽  
Respiratory Syndrome Virus ◽  
Highly Pathogenic

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.

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.

Sign in / Sign up

Export Citation Format

Share Document