Eoin Clancy - Academia.edu (original) (raw)

Papers by Eoin Clancy

Sensors and Actuators B: Chemical, 2021

We report for the first time rotat extraction of total RNA from cell hom novel, solvent-dependent... more We report for the first time rotat extraction of total RNA from cell hom novel, solvent-dependent routing on a c fluidic platform. This system allows f ration and integrated analysis to be i single microfluidic device. Also reporte time is a solvent-triggered valving me performance of on-disc extraction of pur

2017 IEEE Biomedical Circuits and Systems Conference (BioCAS), 2017

We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucle... more We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating, to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range. The motivation for such systems is to implement fast point of care tests in a healthcare setting. Rapid H. Influenzae identification is crucial for efficient treatment to avoid serious health effects (e.g. childhood meningitis). Molecular diagnostic tests offer the sensitivity and speed required for such applications.

Pathology & Oncology Research, 2019

MicroRNAs, as small non-coding regulatory RNAs, play crucial roles in various aspects of breast c... more MicroRNAs, as small non-coding regulatory RNAs, play crucial roles in various aspects of breast cancer biology. They have prognostic and diagnostic value, which makes them very interesting molecules to investigate. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is the gold standard method to analyse miRNA expression in breast cancer patients. This study investigated two RT-qPCR methods (absolute and relative) to determine the expression of ten miRNAs in whole blood samples obtained from luminal A breast cancer patients compared to healthy controls. Whole blood samples were collected from 38 luminal A breast cancer patients and 20 healthy controls in Paxgene blood RNA tubes. Total RNA was extracted and analysed by relative and absolute RT-qPCR. For relative RT-qPCR, miR-16 was used as an endogenous control. For absolute RT-qPCR, standard curves were generated using synthetic miRNA oligonucleotides to determine the absolute copy number of each miRNA. Of the ten miRNAs that were analysed, the absolute RT-qPCR method identified six miRNAs (miR-16, miR-145, miR-155, miR-451a, miR-21 and miR-486) that were upregulated and one miRNA (miR-195) that was downregulated. ROC curve and AUC analysis of the data found that the combination of three miRNAs (miR-145, miR-195 and miR-486) had the best diagnostic value for luminal A breast cancer with an AUC of 0.875, with 76% sensitivity and 81% specificity. On the other hand, the relative RT-qPCR method identified two miRNAs (miR-155 and miR-486) that were upregulated and miR-195, which was downregulated. Using this approach, the combination of three miRNAs (miR-155, miR-195 and miR-486) was showed to have an AUC of 0.657 with 65% sensitivity and 69% specificity. We conclude that miR-16 is not a suitable normalizer for the relative expression profiling of miRNAs in luminal A breast cancer patients. Compared to relative quantification, absolute quantification assay is a better method to determine the expression level of circulating miRNAs in Luminal A breast cancer.

Analytical biochemistry, 2018

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology... more Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA as...

International journal of molecular sciences, Feb 9, 2018

Bacterial meningitis infection is a leading global health concern for which rapid and accurate di... more Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: , and . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for , and of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive sa...

Analytical and bioanalytical chemistry, 2017

The detection and profiling of microRNAs are of great interest in disease diagnosis and prognosis... more The detection and profiling of microRNAs are of great interest in disease diagnosis and prognosis. In this paper, we present a method for the rapid amplification-free detection of microRNAs from total RNA samples. In a two-step sandwich assay approach, fluorescently labeled reporter probes were first hybridized with their corresponding target microRNAs. The reaction mix was then added to a microarray to enable their specific capture and detection. Reporter probes were Tm equalized, enabling specificity by adjusting the length of the capture probe while maintaining the stabilizing effect brought about by coaxial base stacking. The optimized assay can specifically detect microRNAs in spiked samples at concentrations as low as 1 pM and from as little as 100 ng of total RNA in 2 h. The detection signal was linear between 1 and 100 pM (R(2) = 0.99). Our assay data correlated well with results generated by qPCR when we profiled a select number of breast cancer related microRNAs in a total...

Sensors and Actuators B: Chemical, 2017

We present a centrifugal microfluidic "Lab-on-a-Disc" (LoaD) system capable of implementing nucle... more We present a centrifugal microfluidic "Lab-on-a-Disc" (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 10 2-10 4 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.

Journal of microbiological methods, Aug 16, 2016

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) a... more Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the...

BMC infectious diseases, Jan 29, 2015

Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction o... more Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent. Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated...

PLOS ONE, 2015

Data Availability Statement: The data discussed in this publication are deposited in NCBI's Gene ... more Data Availability Statement: The data discussed in this publication are deposited in NCBI's Gene Expression Omnibus (Edgar et al 2002) and are accessible through GEO Series accession number GSE72080 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE72080) (Line 331-334).

Diagnostic Microbiology and Infectious Disease, 2015

ABSTRACT We report for the first time rotationally controlled extraction of total RNA from cell h... more ABSTRACT We report for the first time rotationally controlled extraction of total RNA from cell homogenate through novel, solvent-dependent routing on a centrifugal micro-fluidic platform. This system allows for sample preparation and integrated analysis to be incorporated on a single microfluidic device. Also reported here for the first time is a solvent-triggered valving mechanism and the performance of on-disc extraction of purified total RNA.

Microfluidics and Nanofluidics, 2014

Sensors and Actuators B: Chemical, 2007

Rapid and large sample volume processing capabilities are required for many clinical and environm... more Rapid and large sample volume processing capabilities are required for many clinical and environmental point of care genetic diagnostics scenarios. In this paper we describe the development of a silicon monolithic device for high flow rate DNA extraction. The silicon presents silica surfaces and with chaotropic salt solutions can be used for solid phase extraction. The microfluidic device was designed

Journal of Industrial Microbiology & Biotechnology, 2013

High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in h... more High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 103 cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) a...

Biosensors and Bioelectronics, 2012