Key Performance Indicators: A Framework for Allied Healthcare Educational Institutions.

Background: Performance evaluation in the allied healthcare education sector is complex, making it essential for policymakers and managers to approach it comprehensively and thoughtfully to understand their performance. Hence, the development and monitoring of Key Performance Indicators (KPIs) in this domain must be considered one of the key priorities for the policymakers in AHIs. Aim: This study aims to develop a framework for the AHIs to extract and profile the indicators, measure, and report the results appropriately. Methods: The authors adopted a general review of the literature approach to study the primary goals of the institutional KPI framework, emphasizing the need for benchmarking while implementing KPIs and how to track performance using a KPI dashboard. Results: The study provides the scope, relevant KPI categories, and a list of KPIs for evaluating the effectiveness of allied healthcare programs. The study findings also emphasized the need for benchmarking the KPIs and establishing a KPI dashboard while measuring and monitoring performance. Conclusion: KPIs are considered an invaluable tool that contributes immensely to the performance monitoring process of AHIs, irrespective of the specialties. This helps to identify and guide AHIs for developing KPIs and the associated minimum data set to measure organizational performance and monitor the quality of teaching and learning. In addition, the KPI framework reported in this study is a tool to assist performance monitoring that can subsequently contribute to the overall quality of AHIs.

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Materials and Methods: Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. Results: We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/µL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Discussion: Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

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

Background: Due to their prevalence worldwide, the ß-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. Methods: In this study, three rapid diagnostic single-plex and a prototype triplex assay were developed, using recombinase polymerase amplification 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 samples (n = 18). Results: The RPA-LF assays were able to detect bla CXT-M-group-1, bla CTX-M-group-9 and bla CMY-2-type variants with high sensitivity (82.1%-100%) and specificity (100%) within a short turnaround time (15-20 min for amplification and detection). Conclusions: 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.

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

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/µL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/µL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE 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 sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.

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

BACKGROUND: 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. METHODS: We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity. RESULTS: 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. CONCLUSION: 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 samples 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.

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

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 sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample 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 sample 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.

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

OBJECTIVES: To develop instrument-free point-of-care methods using recombinase polymerase amplification (RPA) technology coupled with a simple lateral flow detection system to detect Neisseria gonorrhoeae and susceptibility to ciprofloxacin. METHODS: 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 amplification test (NAAT)-positive clinical sample extracts (n = 40). RESULTS: 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 sample 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 samples confirmed to have WT gyrA sequences based on Sanger sequencing. CONCLUSIONS: 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.

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

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 individuals. 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.

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

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 amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample 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 TCID50/mL (100-200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample 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.

The JAK inhibitor baricitinib inhibits oncostatin M induction of proinflammatory mediators in ex-vivo synovial derived cells.

OBJECTIVES: To investigate the ex vivo effect of the JAK1/2 inhibitor baricitinib on expression of pro-inflammatory mediators in rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) stimulated with TNFα, IL-1ß and oncostatin M (OSM), and in RA synovial membrane cells (SMCs). METHODS: RA and osteoarthritis (OA) SMCs, were isolated from arthroplasty specimens of RA (n=8) and OA (n=8) patients, respectively, using enzymatic digestion followed by cell propagation to obtain RA (n=5) and OA (n=3) FLS. Normal FLS and normal human foreskin fibroblasts (HSF) were purchased from commercial sources. Fibroblasts were stimulated with cytokines with or without baricitinib. RA SMCs were cultured in the presence of baricitinib without stimulation. JAK/STAT activation and levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, RANTES and IP-10) were determined by qPCR, ELISA and MSD. RESULTS: Baricitinib inhibited OSM-induced JAK signalling in RA synovial fibroblasts and effectively suppressed subsequent expression of the proinflammatory mediators IL-6, MCP-1 and IP-10. However, baricitinib was not effective in altering levels of spontaneously released TNFα, IL-6 and IL-8 in RA SMC. Although both TNFα and IL-1ß signal independently of the JAK/STAT pathway, in HSF, but not in RA FLS, baricitinib significantly inhibited TNFα- and IL-1ß-induced MCP-1 and IP-10 protein levels in a dose dependent manner. Furthermore, baricitinib did not inhibit TNFα- and IL-1ß-induced expression of IL-6, IL-8 and MCP-1 in RA FLS. CONCLUSIONS: These findings are consistent with known signalling pathways employed by OSM, TNFα and IL-1ß, but our data suggest that in HSF, baricitinib may have anti-inflammatory effects via downstream modulation of cytokines and chemokines produced in response to TNFα or IL-1ß.

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

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 (<20 µM), safe (20-100 µM), and toxic (>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.

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

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 sample preparation step, a single-temperature isothermal amplification, 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 individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually 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 detection of NDM and VIM carbapenemase encoding genes by recombinase polymerase amplification and lateral flow-based detection.

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 blaNDM-type and blaVIM-type carbapenemase genes using recombinase polymerase amplification (RPA) combined with a lateral flow detection. The tests could provide results in approximately 15 min when using DNA extracts, with limits of detection of 9.2 copies/µl for the blaNDM-type assay and 7.5 copies/µl for blaVIM-type assay, and successfully detected all isolates harbouring the carbapenemase encoding genes in a panel of 57 isolates. These RPA tests may be suitable for use in low-resource settings to tailor rapid implementation of infection control precautions and antibiotic stewardship.

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

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.

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

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

Repeated Reuse of Deoxyribozyme-Based Logic Gates.

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.

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

Nucleic acid analysis has become highly relevant for point-of-care (POC) diagnostics since the advent of isothermal amplification methods that do not require thermal cycling. In particular, recombinase polymerase amplification (RPA) combined with lateral flow detection offers a rapid and simple solution for field-amenable low-resource nucleic acid testing. Expanding POC nucleic acid tests for the detection of multiple analytes is vital to improve diagnostic efficiency because increased multiplexing capacity enables higher information density combined with reduced assay time and costs. Here, we investigate expanding RPA POC detection by identifying a generic multiplex RPA format that can be combined with a generic multiplex lateral flow device (LFD) to enable binary and molecular encoding for the compaction of diagnostic data. This new technology relies on the incorporation of molecular labels to differentiate nucleic acid species spatially on a lateral flow membrane. In particular, we identified additional five molecular labels that can be incorporated during the RPA reaction for subsequent coupling with LFD detection. Combined with two previously demonstrated successful labels, we demonstrate potential to enable hepta-plex detection of RPA reactions coupled to multiplex LFD detection. When this hepta-plex detection is combined with binary and molecular encoding, an intuitive 7-segment output display can be produced. We note that in all experiments, we used an identical DNA template, except for the 5' label on the forward primer, to eliminate any effects of nucleic acid sequence amplification bias. Our proof-of-concept technology demonstration is highly relevant for developing information-compact POC diagnostics where space and time are premium commodities.

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

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 amplification 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. Sample 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 samples, 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.

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

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 sample volumes were analysed, and 1 PFU per 10 mL where 10 mL sample 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 samples and surface water samples, mean differences in results of the QP assay and the conventional plaque assay were not statistically significant (mean difference ≤ 0.15 log10 PFU/L and 0.5 PFU/10 mL respectively, P > 0.05). The QP is a valuable tool for assessing microbial water quality, which may assist in improving the management of water resources.