A novel cassette method for probe evaluation in the designed biochips

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip

Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 7 10−11 M (190 amol), equivalent to 8.67 7 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 \ub0C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need

One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation

<div><h3>Background</h3><p>In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.</p> <h3>Methodology</h3><p>To address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.</p> <h3>Results and Conclusions</h3><p>We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.</p> </div

Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP

In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions

A multiplexed human papillomavirus (HPV) 16 and 18 diagnostic for cervical cancer screening

Cervical cancer is a major problem in the developing world and low- resource settings where standard screening techniques are not accessible. Cervical cancer is one of the few cancers that can be successfully treated when detected early. Therefore, there exists a high clinical need to screen for cervical cancer early. The etiological agent of cervical cancer is the human papillomavirus (HPV), with 70% of cases related to HPV genotypes 16 and 18. I sought to increase access to screening by developing a fully integrated and multiplexed molecular diagnostic assay to extract, amplify, detect, and distinguish HPV16 and HPV18 DNA on a low-cost paperfluidic platform for point- of-care (POC) applications. Isothermal (one temperature) loop-mediated amplification (LAMP) was used to amplify HPV DNA instead of the traditional polymerase chain reaction (PCR) that requires multiple temperatures. The amplified HPV16 and HPV18 DNA were differentially detected on a simple lateral flow strip – similar to that used in a pregnancy test – generating a visible colorimetric readout for each specific genotype. LAMP amplification is difficult to characterize and current methods were insufficient in providing specificity at the level needed for a multiplexed assay. Therefore, a novel characterization strategy was developed based on fluorescence to distinguish positive LAMP amplification products. This workflow used differential fluorescent tags to identify whether HPV16 DNA or HPV18 DNA was present and simplified complex nonspecific LAMP smears to a specific band pattern. After singleplex HPV16 and HPV18 LAMP assays were optimized with the new workflow, the two singleplex assays were successfully combined into one multiplex reaction with 12 primers, a nontrivial feat. Each assay step – DNA extraction, amplification, and detection – was optimized and integrated into a single chip that can control the timing of each step. Several chip configurations were tested to determine the optimal chip form factor, and a small subset of clinical samples were tested to demonstrate feasibility in low-resource settings. With this diagnostic platform, asymptomatic patients positive for HPV16 DNA and HPV18 DNA can be screened more closely, allocating precious resources to those most at risk, a beneficial use in both low-resource settings and the USA

Microfluidics for Molecular Measurements and Quantitative Distributable Diagnostics

A major challenge in global health care is a lack of portable and affordable quantitative diagnostic devices. This is because classic quantification of biomolecules is typically performed using kinetic assays that require strict control only found in controlled laboratory environments. By using the power of microfluidics, quantitative assays can be performed robustly in a "digital" format that is decoupled from precise kinetics through highly parallelized qualitative reactions. The benefits of performing quantitative assays in a digital format extend beyond just assay robustness to reduction of instrumental complexity, increase in quantitative precision, and an increase in the amount of information that can be gained from a single experiment. These microfluidic architectures, however, are not limited to usage in scenarios of quantification of biomolecules. These architectures can also potentially be extended to answering complex biological questions in single cells, such as determining the 3-dimensional organization of nuclear DNA and RNA

Evaluation of Diagnostic Tests for HIV Detection in Children in Malawi

Annually >40,000 Malawian babies are exposed to HIV, and >1800 get infected. Maternal antibodies limit the utility of HIV antibody testing in children <18 months, and HIV-DNA-PCR is preferred. However, HIV-PCR testing sites are few, PCR is expensive and PCR result turn-around-times (TAT) average 3 months leading to ~ 1/3rd of tested infants being lost to follow-up. A point-of-care HIV Test (POCT) may improve testing uptake and infant retention. This study aimed to identify an appropriate POCT for HIV diagnosis in children <18 months of age and to estimate its impact on infant diagnosis and linkage to HIV care. We conducted mixed methods study in two hospitals in Southern Malawi and evaluated: (1) performance, TAT, usability, acceptability, cost and cost-effectiveness for XpertHIV and compared it with HIV-DNA-PCR, and (2) performance and accuracy of plasma IP-10 in screening for HIV diagnosis and treatment failure among children aged 0-14 years; and (3) explored the potential role of HIV-LAMP assay in the diagnosis of HIV. XpertHIV had high sensitivity (97.8%) and specificity (98.1%). TAT was reduced from 24 days to 5.3 hours: 85% of the children-initiated ART on the day of testing. Compared with qPCR cost ($66.66), XpertHIV was cheaper at$42.34 per diagnostic test. With the difference in test costs, if XpertHIV is adopted nationwide for testing of 40,000 HIV exposed babies, Malawi could save ~USD2,193,538.88 annually. XpertHIV was acceptable to care givers, parents, laboratory technologists and nurses. Although IP-10 levels were higher in those with high viral load and under two years of age, it could not distinguish HIV from other infections. The HIV LAMP optimisation was unsuccessful. XpertHIV, if deployed in Malawi, could improve the diagnosis of paediatric HIV and its treatment uptake, and reduce costs of early infant diagnosis (EID). IP-10 is not an accurate screening marker for HIV in children. Further studies are needed to evaluate whether HIV LAMP is a suitable test for EI

Multiplexed label-free electrical detection of DNA amplification using field effect transistors

The objective of this research project was to develop a miniaturized DNA amplification biosensor for the detection and identification of pathogenic bacteria. Using tailored loop-mediated isothermal amplification (LAMP) and field effect transistors, we developed a microchip platform for multiplexed screening of samples querying the presence of multiple pathogenicity genes. In our platform, ion-sensitive field effect transistors (ISFETs) detect the incorporation of nucleotides during LAMP by monitoring changes in the solution's acidity. Employing transistors as biosensors enables label-free detection of the reaction, simple multiplexing, and seamless integration with required electronics for data acquisition. These characteristics of the detection system and protocols that we developed will make genotyping analysis simple and readily available for different applications that would benefit from low cost, portability, and ease-of-use. Here, we present a series of studies performed in three experimental setups that are related to the multiplexed electrical detection of LAMP and culminate in a large ISFET sensor array microchip that monitors DNA amplification reactions. A first chip consisted of 30 nL silicon oxide wells that were prepared with dried nucleic acid primers for multiplexed on-chip amplification. This initial study demonstrated the high specificity and low limit of detection of on-chip parallel LAMP when used for the detection of E.coli O157, S.enterica, L. monocytogenes, and non O157 Shiga-toxin producing E.coli of the `big six' group. Then, a second chip with novel individually addressable dual-gated ISFETs was fabricated in collaboration with Taiwan Semiconductor Manufacturing Company (TSMC). These devices were used to evaluate and optimize their pH sensing ability, develop methods to do label-free detection of LAMP, and study the sensor performance when biased with polypyrrole quasi-reference electrodes. The last platform, that demonstrates the impressive scalability of the semiconductor technology, is a chip with over a million ISFET sensors distributed in a 7x7 mm2 area. The use of on-chip decoding and routing circuits enables the parallel operation of 1024x1024 sensors in an array for massively multiplexed biosensing. In this platform we applied methods and systems developed previously to perform parallel electrical detection of foodborne pathogens by monitoring DNA amplification reactions in micro-chambers of 250 nL detecting down to 25 copies/reaction in less than 60 min. We demonstrate that the intrinsic redundancy of the high density ISFET array enabled clear identification of electrical signals resulting from the amplification reaction. This microchip for the detection of DNA and the related protocols on reaction miniaturization, parallelism, and electrical detection are poised to be the basis of new detection systems that bring the impressive advances of the semiconductor industry into biological applications

Development of diagnostic platform for detection of biological agents and toxic microalgae using isothermal amplification

L'objectiu principal d’aquesta tesis és desenvolupar sensors d’ADN portàtils i fàcils d’utilitzar per analitzar material genètic al lloc on sigui necessari, superant les limitacions de les tecnologies actuals. Per dur-ho a terme, els sensors d’ADN s’han dissenyat integrant en un únic dispositiu dues tecnologies: l’amplificació isotèrmica d’ADN, tant en fase líquida com en fase sòlida, i la tecnologia de microarrays. A la tesis es detallen els resultats obtinguts per assolir els objectius específics, que inclouen: el desenvolupament d’una plataforma d’anàlisi d’ADN en fase líquida per a la detecció simultània de diversos organismes. L’ús d’amplificació isotèrmica d’ADN en superfície (fase sòlida), combinant tant amb detecció òptica com detecció electroquímica per a la detecció de mostres reals. L’estudi i optimització de la química de superfície dels sensors d’ADN en fase sòlida, i finalment, l’amplificació isotèrmica d’ADN d’algues tòxiques emprant dNTPs modificats amb molècules redox, fet que permet mesurar directament l’ADN amplificat reduint el temps total d’anàlisi.El objetivo principal de la tesis es desarrollar sensores de ADN portátiles y fáciles de utilizar para analizar material genético allí donde sea requerido, superando las limitaciones de las tecnologías actuales. Para llevarlo a cabo, los sensores de ADN se han diseñado integrando en un único dispositivo dos tecnologías: la amplificación isotérmica de ADN tanto en fase líquida como en fase sólida, y la tecnología de microarrays. En la tesis se detallan los resultados obtenidos para lograr los objetivos específicos, que incluyen: el desarrollo de una plataforma de análisis de ADN en fase líquida para la detección simultánea de distintos organismos. El uso de amplificación isotérmica de ADN en superficie (fase sólida) combinado tanto con detección óptica como con detección electroquímica para la detección de muestras reales. El estudio y optimización de la química de superficie de los sensores de ADN en fase sólida, y finalmente, la amplificación isotérmica de ADN de algas tóxicas utilizando dNTPs modificados con moléculas redox, cosa que permite medir el ADN amplificado directamente, reduciendo así el tiempo tota de análisis.The main goal of this doctoral thesis is to present alternative approaches in the field of DNA biosensors, design and develop new isothermal amplification protocols compatible with a portable, easy-to use device that can be deployed for analysis of genetic material at the point-of-care/need, while overcoming some of the currently existing limitations. In order to achieve this objective, a variety of different strategies of effective solid-phase immobilisation and liquid-phase and solid-phase isothermal enzymatic amplification have been studied to achieve lower detection limits with rapid and easy to carry out assays. This work presents a convenient, flexible solution for detecting DNA with biosensors, exploiting a general concept of liquid-phase and solid-phase isothermal amplification and detection, thus integrating two nucleic acid tests, PCR and microarrays, in one single device. The thesis report the work performed to achieve the specific objectives of this doctoral thesis: the development of multiplexing platform for simultaneous detection of several targets, the use of solid-phase recombinase polymerase amplification strategy for DNA amplification with optical and electrochemical detection, the detection of real samples, the study of the surface chemistry and the combination of isothermal amplification with redox labelled dNTPs for the amplification and detection of toxic microalgae as an innovative method that permits incorporation of labels throughout the amplification process facilitating direct electrochemical detection of the DNA products and an inherent shortening of assay time

Buruli Ulcer

A major objective of this open access book is to summarize the current status of Buruli Ulcer (BU) research for the first time. It will identify gaps in our knowledge, stimulate research and support control of the disease by providing insight into approaches for surveillance, diagnosis, and treatment of Buruli Ulcer. Book chapters will cover the history, epidemiology diagnosis, treatment and disease burden of BU and provide insight into the microbiology, genomics, transmission and virulence of Mycobacterium ulcerans. ; Supports further investigation by summarizing state of the art in the field of Buruli ulcer research Enriches understanding of epidemiology of Buruli ulcer in different geographic regions Reviews exhaustively the characteristics of Mycobacterium ulcerans diseas