Joanne Macdonald

Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits

Publisher: American Chemical Society (ACS)

Date: 26-04-2005

DOI: 10.1021/JA043003A

Abstract: A complete set (YES, NOT, AND, and ANDNOT) of molecular scale logic gates based on ligase deoxyribozymes was constructed. The activity of these gates was visualized through the formation of cascades with downstream phosphodieseterase YES gates, which performed fluorogenic cleavage.

Visual Displays that Directly Interface and Provide Read‐Outs of Molecular States via Molecular Graphics Processing Units

Publisher: Wiley

Date: 09-07-2014

DOI: 10.1002/ANIE.201402698

Abstract: The monitoring of molecular systems usually requires sophisticated technologies to interpret nanoscale events into electronic-decipherable signals. We demonstrate a new method for obtaining read-outs of molecular states that uses graphics processing units made from molecular circuits. Because they are made from molecules, the units are able to directly interact with molecular systems. We developed deoxyribozyme-based graphics processing units able to monitor nucleic acids and output alphanumerical read-outs via a fluorescent display. Using this design we created a molecular 7-segment display, a molecular calculator able to add and multiply small numbers, and a molecular automaton able to diagnose Ebola and Marburg virus sequences. These molecular graphics processing units provide insight for the construction of autonomous biosensing devices, and are essential components for the development of molecular computing platforms devoid of electronics.

Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: preparing for the future

Publisher: Springer Science and Business Media LLC

Date: 23-04-2018

DOI: 10.1186/S12985-018-0985-8

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

Publisher: Royal Society of Chemistry (RSC)

Date: 2019

DOI: 10.1039/C8AN01621F

Abstract: Nucleic acid lification has permeated every field in the life sciences since the introduction of the classic polymerase chain reaction (PCR) method in 1983. Yet, despite its fundamental reach, PCR has been constrained within the walls of a laboratory, due to its requirement for a sophisticated thermocycling machine, limiting external application in low-resource settings. New isothermal lification strategies are seeking to break through traditional laboratory boundaries by providing nucleic acid replication at constant temperatures. Of these methods, recombinase polymerase lification (RPA) is one of the fastest developing, experiencing rapid uptake and market, even though it was introduced comparatively late. Critically, RPA's technology potentiates highly accessible and sensitive nucleic acid lification outside of laboratory, and even self-testing. Here we provide a comprehensive review of the equipment-free simplicity of RPA over its first decade of development. Our review includes key knowledge of RPA technology, such as its reaction components, mechanism, sensitivities and specificities, and distinctive detection methods. The review also provides know-how for developing RPA assays, and information about commercially available RPA reaction kits and accessories. We summarise critical RPA experimental tips and issues available through data mining the published literature, to assist researchers in mastering the RPA reaction. We also outline influential hotspots of RPA development, and conclude with outlooks for future development and implications for eclipsing PCR and further revolutionising the life sciences.

Non-viable human embryos as a source of viable cells for embryonic stem cell derivation

Publisher: Elsevier BV

Date: 2009

DOI: 10.1016/S1472-6483(10)60270-2

Abstract: Human embryonic stem cells (hESC) hold great promise for use in regenerative medicine. However, the extraordinary potential of hESC as therapeutic tools is tempered by ethical, moral and political issues surrounding their derivation from human embryos. It has previously been proposed that ethical criteria applied to essential organ donation could be employed for derivation of hESC from irreversibly arrested, and thus organismically dead, human embryos produced during routine IVF procedures. Here, it is shown that arrested embryos do not resume normal development during extended culture, yet most of them contain a substantial number of living cells on embryonic day 6 (72% have <1 viable cell, 47% have <5 viable cells), suggesting that this class of non-viable embryos could be a rich source of viable cells for derivation of hESC lines.

Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.BIOS.2016.04.021

Abstract: Lateral flow biosensors are a leading technology in point-of-care diagnostics due to their simplicity, rapidness and low cost. Their primacy in this arena continues through technological breakthroughs such as multiplexing: the detection of more than one biomarker in a single assay. Multiplexing capacity is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases and reducing diagnostic cost. Here we review, for the first time, the various types and strategies employed for creating multiplexed lateral flow biosensors. These are classified into four main categories in terms of specific application or multiplexing level, namely linear, parameter, spatial and conceptual. We describe the practical applications and implications for each approach and compare their advantages and disadvantages. Importantly, multiplexing is still subject to limitations of the traditional lateral flow biosensor, such as sensitivity and specificity. However, by pushing the limitations of the traditional medium into the multiplex arena, several technological breakthroughs are emerging with novel solutions that further expand the utility of lateral flow biosensing for point-of-care applications.

Evaluation of three rapid low-resource molecular tests for Nipah virus

Publisher: Frontiers Media SA

Date: 09-02-2023

DOI: 10.3389/FMICB.2022.1101914

Abstract: Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal lification coupled with lateral flow detection. These tests include a simple and fast one-step s le processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/μL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000–100,000 TCID 50 /mL (100–200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiV B ) and Malaysia (NiV M ), and took 30 min from s le to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices

Publisher: American Society for Microbiology

Date: 14-02-2023

DOI: 10.1128/SPECTRUM.02796-22

Abstract: The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical s le matrices.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes [version 2; peer review: 2 approved]

Publisher: F1000 Research Ltd

Date: 22-12-2022

DOI: 10.12688/GATESOPENRES.13534.2

Abstract: The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel s le preparation step, a single-temperature isothermal lification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both in idually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With in idually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1 n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48–100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48–100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes

Publisher: F1000 Research Ltd

Date: 19-07-2022

DOI: 10.12688/GATESOPENRES.13534.1

Abstract: The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel s le preparation step, a single-temperature isothermal lification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both in idually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With in idually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1 n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48–100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48–100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

Translational control of enzyme scavenger expression with toxin-induced micro RNA switches

Publisher: Springer Science and Business Media LLC

Date: 28-01-2021

DOI: 10.1038/S41598-021-81679-6

Abstract: Biological computation requires in vivo control of molecular behavior to progress development of autonomous devices. miRNA switches represent excellent, easily engineerable synthetic biology tools to achieve user-defined gene regulation. Here we present the construction of a synthetic network to implement detoxification functionality. We employed a modular design strategy by engineering toxin-induced control of an enzyme scavenger. Our miRNA switch results show moderate synthetic expression control over a biologically active detoxification enzyme molecule, using an established design protocol. However, following a new design approach, we demonstrated an evolutionarily designed miRNA switch to more effectively activate enzyme activity than synthetically designed versions, allowing markedly improved extrinsic user-defined control with a toxin as inducer. Our straightforward new design approach is simple to implement and uses easily accessible web-based databases and prediction tools. The ability to exert control of toxicity demonstrates potential for modular detoxification systems that provide a pathway to new therapeutic and biocomputing applications.

Lessons for improved COVID-19 surveillance from the scale-up of malaria testing strategies

Publisher: Springer Science and Business Media LLC

Date: 20-07-2022

DOI: 10.1186/S12936-022-04240-4

Abstract: Effective control of infectious diseases is facilitated by informed decisions that require accurate and timely diagnosis of disease. For malaria, improved access to malaria diagnostics has revolutionized malaria control and elimination programmes. However, for COVID-19, diagnosis currently remains largely centralized and puts many low- and middle-income countries (LMICs) at a disadvantage. Malaria and COVID-19 are infectious diseases that share overlapping symptoms. While the strategic responses to disease control for malaria and COVID-19 are dependent on the disease ecologies of each disease, the fundamental need for accurate and timely testing remains paramount to inform accurate responses. This review highlights how the roll-out of rapid diagnostic tests has been fundamental in the fight against malaria, primarily within the Asia Pacific and along the Greater Mekong Subregion. By learning from the successful elements of malaria control programmes, it is clear that improving access to point-of-care testing strategies for COVID-19 will provide a suitable framework for COVID-19 diagnosis in not only the Asia Pacific, but all malarious countries. In malaria-endemic countries, an integrated approach to point-of-care testing for COVID-19 and malaria would provide bi-directional benefits for COVID-19 and malaria control, particularly due to their paralleled likeness of symptoms, infection control strategies and at-risk in iduals. This is especially important, as previous disease pandemics have disrupted malaria control infrastructure, resulting in malaria re-emergence and halting elimination progress. Understanding and combining strategies may help to both limit disruptions to malaria control and support COVID-19 control.

New therapeutic approaches and novel alternatives for organophosphate toxicity

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.TOXLET.2018.03.028

Synthetic Biology Provides a Toehold in the Fight against Zika

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.CHOM.2016.05.020

Abstract: The World Health Organization has identified the development of a rapid point-of-care diagnostic test for Zika virus as a top research priority. In a recent Cell paper, Pardee et al. (2016) have exploited recent advances in synthetic biology and molecular engineering to deliver a sensitive, affordable, and field-ready Zika test.

Development of Trainable Deoxyribozyme-Based Game Playing Automaton

Publisher: Humana Press

Date: 2012

DOI: 10.1007/978-1-61779-545-9_26

Abstract: Molecular automata are self-operating machines serially exchanging information with their environment while changing their configurations. Molecular protoautomata are devices trained in a series of sessions with an operator to become molecular automata. Reconfigurable deoxyribozyme-based logic gates can be used to build multipurpose protoautomata, reprogrammable devices that go beyond single-purpose "hard-wired" molecular automata. Molecular array of YES and AND gates (MAYA)-III is such a protoautomaton that can be taught by ex le to play a game. Training of MAYA-III is a process that does not require the operator to be familiar with the underlying molecular programming. Herein we provide the instructions to construct this protoautomaton, with particular focus on the optimization of computing components.

Rapid detection of kdr mutation F1534C in Aedes aegypti using recombinase polymerase amplification and lateral flow dipsticks

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.PESTBP.2022.105209

Abstract: Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming s le processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step s le preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes. We trialled the rapid F1534C pyrethroid resistance test using insecticide resistant Ae. aegypti mosquito bodies and compared results to a conventional, allele-specific quantitative PCR (AS-qPCR) coupled with melt curve genotyping in corresponding mosquito heads. From a strain of Ae. aegypti established from an insecticide resistant population in Merida, Mexico (n = 27), all the mosquito bodies (n = 27) tested positive with the rapid F1534C test regardless of whether they were homozygous or heterozygous. To assess diagnostic test specificity, we confirmed that F1534 was not detected in laboratory-reared, fully susceptible Ae. aegypti mosquito bodies (n = 28) using the rapid F1534C test or the conventional AS-qPCR melt curve analysis. All corresponding mosquito heads (n = 28) were homozygous wild-type FF1534. The rapid F1534C test thus demonstrated 100% diagnostic sensitivity (95% CI: 87.23% to 100%) and 100% diagnostic specificity (95% CI: 87.66% to 100.00%) for detection of the F1534C pyrethroid resistant single nucleotide polymorphism (SNP) in both heterozygous and homozygous Ae. aegypti. In the collection of mutant mosquitoes from Mexico, CC1534 homozygous mutants occurred at a frequency of 74.1% (n = 20) and FC heterozygous mutants at a frequency of 25.9% (n = 7). The rapid F1534C test significantly reduced the s le processing and testing time from approximately 6 h for the AS-qPCR melt curve analysis to only 25 min. These results demonstrate significant potential for our approach to resistance testing as a field-based, low-resource, rapid alternative to time-consuming and expensive laboratory-based detection.

Correction: Review: a comprehensive summary of a decade development of the recombinase polymerase amplification.

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/C9AN90127B

Abstract: Correction for 'Review: a comprehensive summary of a decade development of the recombinase polymerase lification' by Jia Li et al., Analyst, 2019, 144, 31-67.

Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder

Publisher: American Chemical Society (ACS)

Date: 2006

DOI: 10.1021/BI051871U

Abstract: We have developed an array of seven deoxyribozyme-based molecular logic gates that behaves as a full adder in a single solution, with three oligonucleotides as inputs and two independent fluorogenic cleavage reactions as carry and sum outputs. The sum output consisted of four new deoxyribozyme-based logic gates: an ANDAND gate and three ANDNOTANDNOT gates. These gates required the design of a generic three-input deoxyribozyme-based logic gate that can use any three-way combination of activating or inactivating inputs. This generic gate design utilizes an additional inverting element that hybridizes to convert YES logic into NOT logic and vice versa. The system represents the first solution-phase, single test tube, enzymatic full adder and shows the complexity of control over molecular scale events that can be achieved with deoxyribozyme-based logic gates. Similar systems could be applied to control autonomous therapeutic and diagnostic devices.

Proof-of-concept, rapid, instrument-free molecular detection of Neisseria gonorrhoeae and ciprofloxacin susceptibility

Publisher: Oxford University Press (OUP)

Date: 26-07-2022

DOI: 10.1093/JAC/DKAC242

Abstract: To develop instrument-free point-of-care methods using recombinase polymerase lification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. For identification of gonococcal infection, an RPA-based method was developed targeting the gonococcal porA pseudogene (NG-porA-RPA). For ciprofloxacin susceptibility, predictive WT sequences at codons 91 and 95 of the gonococcal gyrA DNase gene were targeted. Given the known complexities of SNP detection using RPA (e.g. the ability to accommodate mismatches) we trialled several different assays incorporating various additional non-template mismatches in the oligonucleotide sequences to reduce affinity for the mutant (resistant) gyrA sequences. Assays were evaluated using a bank of N. gonorrhoeae (n = 10) and non-gonococcal (n = 5) isolates and a panel of N. gonorrhoeae nucleic acid lification test (NAAT)-positive clinical s le extracts (n = 40). The NG-porA-RPA assay was specific to N. gonorrhoeae and provided a positive percentage agreement (PPA) of 87.5% (35/40) compared with a commercial N. gonorrhoeae NAAT when applied to the 40 clinical s le extracts. For gyrA, the non-template bases successfully reduced banding intensity for double-mutant strains (mutations at both 91 and 95), but not for rarer single-mutant (91 only) strains. The most promising gyrA assay, NG-gyrA-RPA08, correctly detected 83% (25/30) of infections from NAAT-positive clinical s les confirmed to have WT gyrA sequences based on Sanger sequencing. These proof-of-concept data show that RPA technology has considerable promise for detecting N. gonorrhoeae and associated antibiotic susceptibility and would offer a diagnostic-based stewardship strategy identified as urgently needed by the WHO.

Inkjet printing for biosensor fabrication: combining chemistry and technology for advanced manufacturing

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5LC00235D

Abstract: Inkjet printing is emerging at the forefront of biosensor fabrication technologies.

Mechanical properties of silk of the Australian golden orb weaversNephila pilipesandN. plumipes

Publisher: The Company of Biologists

Date: 2018

DOI: 10.1242/BIO.029249

Abstract: Silks from orb-weaving spiders are exceptionally tough, producing a model polymer for biomimetic fibre development. The mechanical properties of naturally-spun silk threads from two species of Australian orb-weavers, Nephila pilipes and N. plumipes, were examined here in relation to overall thread diameter, the size and number of fibres within threads, and spider size. N. pilipes, the larger of the two species, had significantly tougher silk with higher strain capacity than its smaller congener, producing threads with average toughness of 150 MJ m−3, despite thread diameter, mean fibre diameter and number of fibres per thread not differing significantly between the two species. Within N. pilipes, smaller silk fibres were produced by larger spiders, yielding tougher threads. In contrast, while spider size was correlated with thread diameter in N. plumipes, there were no clear patterns relating to silk toughness, which suggests that the differences in properties between the silk of the two species arise through differing molecular structure. Our results support previous studies that found the mechanical properties of silk differ between distantly related spider species, and extends on that work to show that the mechanical and physical properties of silk from more closely related species can also differ remarkably.

Thermostable Variants of Cocaine Esterase for Long-Time Protection against Cocaine Toxicity

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 05-11-2008

DOI: 10.1124/MOL.108.049486

A DNA Logic Gate Automaton for Detection of Rabies and Other Lyssaviruses

Publisher: Wiley

Date: 03-05-2017

DOI: 10.1002/CPHC.201700072

Abstract: Immediate activation of biosensors is not always desirable, particularly if activation is due to non-specific interactions. Here we demonstrate the use of deoxyribozyme-based logic gate networks arranged into visual displays to precisely control activation of biosensors, and demonstrate a prototype molecular automaton able to discriminate between seven different genotypes of Lyssaviruses, including Rabies virus. The device uses novel mixed-base logic gates to enable detection of the large ersity of Lyssavirus sequence populations, while an ANDNOT logic gate prevents non-specific activation across genotypes. The resultant device provides a user-friendly digital-like, but molecule-powered, dot-matrix text output for unequivocal results read-out that is highly relevant for point of care applications.

Molecular Automata

Publisher: Springer New York

Date: 2012

DOI: 10.1007/978-1-4614-1800-9_123

Corrigendum to: “Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses” [J. Biosens. Bioelectron. (2016) 177–192]

Publisher: Elsevier BV

Date: 11-2016

DOI: 10.1016/J.BIOS.2016.06.004

Discovery of New Classes of Compounds that Reactivate Acetylcholinesterase Inhibited by Organophosphates

Publisher: Wiley

Date: 09-09-2015

DOI: 10.1002/CBIC.201500348

Rapid sample preparation and low-resource molecular detection of hepatopancreatic parvoviruses (HPV) by recombinase polymerase amplification lateral flow detection assay in shrimps (Fenneropenaeus mer

Publisher: Public Library of Science (PLoS)

Date: 09-11-2022

DOI: 10.1371/JOURNAL.PONE.0276164

Abstract: Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments. We developed a rapid shrimp HPV test that uses (1) a simple three-step s le preparation protocol, followed by (2) isothermal recombinase polymerase lification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp s le matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity. The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F . merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86–100%) and 100% specificity (95% CI: 66–100%) for detection of HPV. The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from s les and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.

Multiplex lateral flow detection and binary encoding enables a molecular colorimetric 7-segment display

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C5LC01323B

Abstract: First proof-of-concept generic 7-segment display for a lateral flow biosensor, with exponential multiplexing detection ability via a binary encoding concept.

Advancing the Deoxyribozyme-Based Logic Gate Design Process

Publisher: Springer Berlin Heidelberg

Date: 2009

DOI: 10.1007/978-3-642-10604-0_5

Detection of a single nucleotide polymorphism for insecticide resistance using recombinase polymerase amplification and lateral flow dipstick detection

Publisher: Elsevier BV

Date: 09-2022

DOI: 10.1016/J.SNB.2022.132085

Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification

Publisher: Oxford University Press (OUP)

Date: 04-08-2015

DOI: 10.1093/PROTEIN/GZV036

Abstract: The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

Core Outcome Measures for Trials in People With Coronavirus Disease 2019: Respiratory Failure, Multiorgan Failure, Shortness of Breath, and Recovery

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 05-01-2021

DOI: 10.1097/CCM.0000000000004817

Abstract: Respiratory failure, multiple organ failure, shortness of breath, recovery, and mortality have been identified as critically important core outcomes by more than 9300 patients, health professionals, and the public from 111 countries in the global coronavirus disease 2019 core outcome set initiative. The aim of this project was to establish the core outcome measures for these domains for trials in coronavirus disease 2019. Three online consensus workshops were convened to establish outcome measures for the four core domains of respiratory failure, multiple organ failure, shortness of breath, and recovery. International. About 130 participants (patients, public, and health professionals) from 17 countries attended the three workshops. None. Respiratory failure, assessed by the need for respiratory support based on the World Health Organization Clinical Progression Scale, was considered pragmatic, objective, and with broad applicability to various clinical scenarios. The Sequential Organ Failure Assessment was recommended for multiple organ failure, because it was routinely used in trials and clinical care, well validated, and feasible. The Modified Medical Research Council measure for shortness of breath, with minor adaptations (recall period of 24 hr to capture daily fluctuations and inclusion of activities to ensure relevance and to capture the extreme severity of shortness of breath in people with coronavirus disease 2019), was regarded as fit for purpose for this indication. The recovery measure was developed de novo and defined as the absence of symptoms, resumption of usual daily activities, and return to the previous state of health prior to the illness, using a 5-point Likert scale, and was endorsed. The coronavirus disease 2019 core outcome set recommended core outcome measures have content validity and are considered the most feasible and acceptable among existing measures. Implementation of the core outcome measures in trials in coronavirus disease 2019 will ensure consistency and relevance of the evidence to inform decision-making and care of patients with coronavirus disease 2019.

Molecular Automata

Publisher: Springer New York

Date: 2009

DOI: 10.1007/978-0-387-30440-3_335

Structural analysis of thermostabilizing mutations of cocaine esterase

Publisher: Oxford University Press (OUP)

Date: 30-04-2010

DOI: 10.1093/PROTEIN/GZQ025

Rapid, sensitive, and specific, low-resource molecular detection of Hendra virus

Publisher: Elsevier BV

Date: 06-2023

DOI: 10.1016/J.ONEHLT.2023.100504

Complexing deoxyribozymes with RNA aptamers for detection of the small molecule theophylline

Publisher: Elsevier BV

Date: 02-2022

DOI: 10.1016/J.BIOS.2021.113774

Abstract: Biointegrative information processing systems offer a great advantage to autonomous biodevices, as their capacity for biological computation provides the ability to sense the state of more complex environments and better integrate with downstream biological regulation systems. Deoxyribozymes (DNAzymes) and aptamers are of interest to such computational biosensing systems due to the enzymatic properties of DNAzymes and the ligand-inducible conformational structures of aptamers. Herein, we describe a novel method for providing ligand-responsive allosteric control to a DNAzyme using an RNA aptamer. We designed a NOT-logic-compliant E6 DNAzyme to be complementary to an RNA aptamer targeting theophylline, such that the aptamer competitively interacted with either theophylline or the DNAzyme, and disabled the DNAzyme only when theophylline concentration was below a given threshold. Out of our seven designed "complexing aptazymes," three demonstrated effective theophylline-responsive allosteric regulation (2.84 ± 3.75%, 4.97 ± 2.92%, and 8.91 ± 4.19% activity in the absence of theophylline 46.29 ± 3.36%, 50.70 ± 10.15%, and 61.26 ± 6.18% activity in the presence of theophylline). Moreover, the same three complexing aptazymes also demonstrated the ability to semi-quantitatively determine the concentration of theophylline present in solution, successfully discriminating between therapeutically ineffective ( 100 μM) theophylline concentrations. Our method of using an RNA aptamer for ligand-responsive allosteric control of a DNAzyme expands the way aptamers can be configured for biosensing, and suggests a pathway for embedding DNAzymes to provide enhanced information processing and control of biological systems.

Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric 7-Segment Display

Publisher: American Chemical Society (ACS)

Date: 07-2019

DOI: 10.1021/ACSOMEGA.9B01097

Technical aspects of using human adenovirus as a viral water quality indicator

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.WATRES.2016.03.042

Abstract: Despite dramatic improvements in water treatment technologies in developed countries, waterborne viruses are still associated with many of cases of illness each year. These illnesses include gastroenteritis, meningitis, encephalitis, and respiratory infections. Importantly, outbreaks of viral disease from waters deemed compliant from bacterial indicator testing still occur, which highlights the need to monitor the virological quality of water. Human adenoviruses are often used as a viral indicator of water quality (faecal contamination), as this pathogen has high UV-resistance and is prevalent in untreated domestic wastewater all year round, unlike enteroviruses and noroviruses that are often only detected in certain seasons. Standard methods for recovering and measuring adenovirus numbers in water are lacking, and there are many variations in published methods. Since viral numbers are likely under-estimated when optimal methods are not used, a comprehensive review of these methods is both timely and important. This review critically evaluates how estimates of adenovirus numbers in water are impacted by technical manipulations, such as during adenovirus concentration and detection (including culturing and polymerase-chain reaction). An understanding of the implications of these issues is fundamental to obtaining reliable estimation of adenovirus numbers in water. Reliable estimation of HAdV numbers is critical to enable improved monitoring of the efficacy of water treatment processes, accurate quantitative microbial risk assessment, and to ensure microbiological safety of water.

Nucleic Acids for Computation

Publisher: Springer New York

Date: 2009

DOI: 10.1007/978-0-387-73711-9_14

Rapid molecular detection of CMY-2, and CTX-M group 1 and 9 variants via recombinase polymerase amplification

Publisher: Oxford University Press (OUP)

Date: 02-03-2023

DOI: 10.1093/JACAMR/DLAD023

Abstract: Due to their prevalence worldwide, the β-lactamases CTX-M and plasmid-mediated CMY-2 are important antimicrobial resistance enzymes in a clinical setting. While culture- and PCR-based detection methods exist for these targets, they are time consuming and require specialist equipment and trained personnel to carry out. In this study, three rapid diagnostic single-plex and a prototype triplex assay were developed, using recombinase polymerase lification with lateral flow detection (RPA-LF), and tested for their sensitivity and specificity using two isolate DNA panels (n = 90 and n = 120 isolates). In addition, the RPA-LF assays were also tested with a small number of faecal extract s les (n = 18). The RPA-LF assays were able to detect blaCXT-M-group-1, blaCTX-M-group-9 and blaCMY-2-type variants with high sensitivity (82.1%–100%) and specificity (100%) within a short turnaround time (15–20 min for lification and detection). RPA-LF assays developed in this study have the potential to be used at or close to the point of care, as well as in low-resource settings, producing rapid results to support healthcare professionals in their treatment decisions.

Enhanced Detection Efficiency of Plastic Scintillators upon Incorporation of Zirconia Nanoparticles

Publisher: MYU K.K.

Date: 2015

DOI: 10.18494/SAM.2015.1090

The QuantiPhage assay: A novel method for the rapid colorimetric detection of coliphages using cellulose pad materials

Publisher: Elsevier BV

Date: 02-2019

DOI: 10.1016/J.WATRES.2018.10.089

Abstract: Assessment of viral contamination is essential for monitoring the microbial quality of water and protection of public health, as human virus presence is not accurately determined using bacterial indicators. Currently, the time required for conventional viral testing means that water contaminated with human pathogens may be used (e.g. for drinking, recreation or irrigation) days before results are available. Here we report a new rapid method for coliphage enumeration, the QuantiPhage (QP) assay. The novelty of the assay is the use of cellulose absorbent pad materials to support coliphage growth and colorimetric detection, in place of agar that is used in the plaque assay. In addition to saving time associated with agar preparation and tempering, the QP assay enabled enumeration of somatic coliphages in 1.5-2 h and F+ coliphages in 2.5-3 h. The assays were highly sensitive, with a lower detection limit of 1 plaque forming unit (PFU) per mL where 1 mL s le volumes were analysed, and 1 PFU per 10 mL where 10 mL s le volumes were analysed. This is the first rapid culture assay to enable low numbers of coliphages to be reliably detected and to produce directly equivalent results to agar-based plaque assays. A novel gelatin-immobilisation method is also reported, that reduces time to prepare bacterial cells from ∼20 h to 40-60 min (depending on the assay format), and provides a ready to use form of cells, that is compatible with rapid detection and kit formats. When applied to analysis of somatic coliphages in wastewater s les and surface water s les, mean differences in results of the QP assay and the conventional plaque assay were not statistically significant (mean difference ≤ 0.15 log

Repeated Reuse of Deoxyribozyme-Based Logic Gates

Publisher: American Chemical Society (ACS)

Date: 16-10-2019

DOI: 10.1021/ACS.NANOLETT.9B02326

Abstract: Deoxyribozymes (DNAzymes) have demonstrated a significant capacity for biocomputing and hold promise for information processing within advanced biological devices if several key capabilities are developed. One required capability is reuse-having DNAzyme logic gates be cyclically, and controllably, activated and deactivated. We designed an oligonucleotide-based system for DNAzyme reuse that could (1) remove previously bound inputs by addition of complementary oligonucleotides via toe-hold mediated binding and (2) diminish output signal through the addition of quencher-labeled oligonucleotides complementary to the fluorophore-bound substrate. Our system demonstrated, for the first time, the ability for DNAzymes to have their activity toggled, with activity returning to 90-125% of original activity. This toggling could be performed multiple times with control being exerted over when the toggling occurs, with three clear cycles observed before the variability in activity became too great. Our data also demonstrated that fluorescent output of the DNAzyme activity could be actively removed and regenerated. This reuse system can increase the efficiency of DNAzyme-based logic circuits by reducing the number of redundant oligonucleotides and is critical for future development of reusable biodevices controlled by logical operations.

Advances in isothermal amplification: novel strategies inspired by biological processes

Publisher: Elsevier BV

Date: 02-2015

DOI: 10.1016/J.BIOS.2014.08.069

Abstract: Nucleic acid lification is an essential process in biological systems. The in vitro adoption of this process has resulted in powerful techniques that underpin modern molecular biology. The most common tool is polymerase chain reaction (PCR). However, the requirement for a thermal cycler has somewhat limited applications of this classic nucleic acid lification technique. Isothermal lification, on the other hand, obviates the use of a thermal cycler because reactions occur at a single temperature. Isothermal lification methods are erse, but all have been developed from an understanding of natural nucleic acid lification processes. Here we review current isothermal lification methods as classified by their enzymatic mechanisms. We compare their advantages, disadvantages, efficiencies, and applications. Finally, we mention some new developments associated with this technology, and consider future possibilities in molecular engineering and recombinant technologies that may develop from an appreciation of the molecular biology of natural systems.

ISO

Publisher: ACM

Date: 08-2011

DOI: 10.1145/2147805.2147859

Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics

Publisher: Informa UK Limited

Date: 30-10-2015

DOI: 10.1586/14737159.2015.1090877

Abstract: Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase lification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid lification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection

Publisher: American Society for Microbiology

Date: 15-11-2005

DOI: 10.1128/JVI.79.22.13924-13933.2005

Abstract: The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Lateral flow biosensors

Publisher: Institution of Engineering and Technology

Date: 19-07-2016

DOI: 10.1049/PBHE001E_CH13

Medium Scale Integration of Molecular Logic Gates in an Automaton

Publisher: American Chemical Society (ACS)

Date: 07-10-2006

DOI: 10.1021/NL0620684

Abstract: The assembly of molecular automata that perform increasingly complex tasks, such as game playing, presents an unbiased test of molecular computation. We now report a second-generation deoxyribozyme-based automaton, MAYA-II, which plays a complete game of tic-tac-toe according to a perfect strategy. In silicon terminology, MAYA-II represents the first "medium-scale integrated molecular circuit", integrating 128 deoxyribozyme-based logic gates, 32 input DNA molecules, and 8 two-channel fluorescent outputs across 8 wells.

Evaluation of MinION nanopore sequencing for rapid enterovirus genotyping

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.VIRUSRES.2018.05.010

Abstract: Enteroviruses (EV) are associated with a range of serious infections, including aseptic meningitis, hand foot and mouth disease, myocarditis, acute flaccid paralysis and encephalitis. Improved methods for assessing EV genotypic ersity could assist molecular epidemiology studies, clinical diagnosis and environmental surveillance. We report new methods for EV genome lification, and subsequent genotyping using the miniaturised MinION sequencing device. Importantly, this next-generation sequencer enabled correct strain-level assignment of identity for the EV-A71 isolate assessed, where strains with up to 99.7% similarity were discriminated. In addition, an accurate consensus sequence was produced for EV-A71 isolate RNA, with 99.3-99.6% similarity to the reference sequence. Thus, the long nanopore reads enabled rapid whole genome sequencing and strain level identification of EV- A71 isolate RNA. We also demonstrated potential for using MinION sequencing for direct detection of EV in water s les, which may have application for ersity analysis, water quality monitoring, and environmental surveillance.

Rapid detection of NDM and VIM carbapenemase encoding genes by recombinase polymerase amplification and lateral flow–based detection

Publisher: Springer Science and Business Media LLC

Date: 11-05-2021

DOI: 10.1007/S10096-021-04267-6

Abstract: Carbapenemase-producing organisms (CPOs) pose a serious clinical threat and rapid detection tools are essential to aid in patient management. We developed rapid and simple molecular tests to detect bla

With great structure comes great functionality: Understanding and emulating spider silk

Publisher: Springer Science and Business Media LLC

Date: 18-12-2014

DOI: 10.1557/JMR.2014.365

DNA Heat Treatment for Improving qPCR Analysis of Human Adenovirus in Wastewater

Publisher: Springer Science and Business Media LLC

Date: 10-04-2017

DOI: 10.1007/S12560-017-9294-4

Rapid assessment of viral water quality using a novel recombinase polymerase amplification test for human adenovirus

Publisher: Springer Science and Business Media LLC

Date: 21-08-2019

DOI: 10.1007/S00253-019-10077-W

Abstract: Sensitive and rapid methods for determining viral contamination of water are critical, since illness can be caused by low numbers of viruses and bacterial indicators do not adequately predict viral loads. We developed novel rapid assays for detecting the viral water quality indicator human adenovirus (HAdV). A simple 15-min recombinase polymerase lification step followed by a 5-min lateral flow detection is used. Species-specific assays were developed to discriminate HAdV A, B, C and F, and combined into a multiplex test (Ad-FAC). Species-specific assays enabled detection of 10-50 copies of the HAdV plasmid. S le testing using methods optimised for wastewater analysis indicated the Ad-FAC assay showed 100% sensitivity and 100% specificity when compared with HAdV qPCR, with a detection limit as low as 50 gene copies. This is the first study to demonstrate the use of RPA for detecting enteric viruses in water s les, to assess virological water quality. The ability to rapidly detect enteric virus contamination of water could assist in more effective management of water safety and better protection of public health.

BioCifer Pty Ltd

Location: Australia

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University Of The Sunshine Coast

Location: Australia

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Columbia University

Location: United States of America

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100179

Start Date: 03-2018

End Date: 12-2019

Amount: $3,189,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100206

Start Date: 05-2017

End Date: 12-2017

Amount: $550,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100119

Start Date: 2014

End Date: 12-2015

Amount: $2,000,000.00

Funder: Australian Research Council