Antimicrobial resistance (AMR) is recognised as one of the greatest scientific challenges of the 21st century, disproportionately affecting people living in low- and middle-income countries. With bacterial pathogens becoming increasingly resistant to antibiotics, there is an urgent need for innovative approaches to combat this growing threat. The World Health Organization has recognised this need and prioritised further research to enhance diagnostics, surveillance and our understanding the epidemiology and drivers of AMR. The Industrial Transformation Research Hub to Combat AMR, or the AMR Hub, is an Australian collaborative private–public research partnership involving over 20 organisations. It aims to foster multidisciplinary collaborations across sectors and develop wholistic solutions that address barriers to the commercialisation of tools to minimise the risks of AMR. The AMR Hub’s research is focusing on sexually transmitted infections, which are increasingly resistant to antibiotics and have few alternative candidates in the pipeline. Investigators are together developing novel diagnostics, optimising treatment, identifying tools to detect active bacterial infections, and engaging stakeholders to optimise AMR innovation. Through a multidisciplinary ecosystem across sectors, the AMR Hub seeks to fast-track the development of adaptable technologies, new antibiotics and stewardship innovations for prevention, while also addressing societal, economic and commercial aspects of AMR solutions.

The probe technology described in this paper facilitates detection and discrimination of multiple targets in a single fluorescent channel during PCR. This provides a strategy for doubling the number of targets that can be analysed simultaneously on existing PCR instruments. These probes are referred to as PlexProbes and produce fluorescence that can be switched ‘on’ or ‘off’ in the presence of target by manipulating the temperature. During PCR, fluorescence can be measured at multiple temperatures allowing discrimination of specific targets at defined temperatures. In a single fluorescent channel, a model duplex assay allowed either real-time or endpoint detection of Chlamydia trachomatis (CT) at 52°C and end-point detection of Neisseria gonorrhoeae (GC) at 74°C. Using this model system, as few as 40 copies of each specific target could be detected as single infection or co-infection, regardless of the presence or absence of the other target. A PlexProbe prototype assay for sexually transmitted infections (PP-STI) which simultaneously enables detection and differentiation of six targets using only three fluorescent channels was then constructed and evaluated. The PP-STI assay detects GC (2 gene targets), CT, Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) and an internal control (IC). To evaluate assay performance, a panel of archived clinical samples (n = 337) were analysed using PP-STI and results compared to those obtained with a commercially available diagnostic assay. The overall agreement between results obtained with the PP-STI assay and the reference test was greater than 99.5%. PlexProbes offer a method of detecting more targets from a single diagnostic test, empowering physicians to make evidence-based treatment decisions while conserving time, labour, sample volume and reagent costs.

The development of Subzymes demonstrates how the catalytic activity of DNAzymes can be controlled for detecting nucleic acids; however, Subzymes alone lack the sensitivity required to detect low target concentrations. To improve sensitivity, we developed a feedback system using a pair of cross-catalytic Subzymes. These were individually tethered to microparticles (MP) and separated by a porous membrane rendering them unable to interact. In the presence of a target, active PlexZymes® cleave a first Subzyme, which separates a first DNAzyme from its MP, allowing the DNAzyme to migrate through the membrane, where it can cleave a second Subzyme. This releases a second DNAzyme which can now migrate through the membrane and cleave more of the first Subzyme, thus initiating a cross-catalytic cascade. Activated DNAzymes can additionally cleave fluorescent substrates, generating a signal, and thereby, indicating the presence of the target. The method detected 1 fM of DNA homologous to the ompA gene of Chlamydia trachomatis within 30 min, demonstrating a 10,000-fold increase in sensitivity over PlexZyme detection alone. The Subzyme cascade is universal and can be triggered by any target by modifying the target sensing arms of the PlexZymes. Further, it is isothermal, protein-enzyme-free and shows great potential for rapid and affordable biomarker detection.

Background Major challenges in the management of infectious diseases include treatment failure due to antimicrobial resistance (AMR) and the lack of a reliable test of cure (TOC). Whilst culture is a trusted method it is slow, and with the widespread use of nucleic acid amplification tests (NAATs), many labs no longer retain culture capabilities. NAATs can assess AMR by detecting microbial mutations associated with resistance; however, this approach requires knowledge of the molecular mechanism(s), and as new mutations emerge, tests need to be reconfigured. Uses of NAATs for TOC is problematic since residual DNA and RNA have been reported following effective therapy. The VITA method provides a new tool which can overcome current drawbacks. Methods The VITA Index is the ratio of the number of copies of a gene and its associated transcripts to those of a non-transcribed region of DNA. It provides a relative measure of active transcription regardless of the quality/quantity of specimen. The approach has several applications. Firstly, following addition of antibiotic to a specimen, e.g. for 5 min/37°C or 15 min/room temperature, Total Nucleic Acid can be amplified by VITA RT-PCR. Comparison of the VITA indices ± drug will relate to antibiotic resistance or sensitivity. VITA RT-PCR can later provide a TOC, where VITA indices of a specimen can fall either above or below a predetermined threshold, indicating a viable or cleared infection respectively. Results Both sensitivity and resistance to different antibiotics has been demonstrated in vitro on Chlamydia trachomatis, with significant decreases in VITA in the presence of drug in sensitive (p<0.05), but not in resistant strains. Further, urine obtained from a patient post-treatment was analysed, and consistent with clinical evidence of ongoing infection, the VITA Index indicated viable chlamydia. Conclusion In conclusion, VITA provides a powerful new approach for rapid determination of AMR and TOC. Disclosure No significant relationships.

Rapid, sensitive and affordable nucleic acid sensors that can operate in a point of care (POC) setting are highly desired; however, their availability and implementation remain limited. We report the development of a strategy for building sensors which use novel catalytic nucleic acid structures, herein referred to as 'Subzymes'. We examine the effectiveness of Subzymes to mediate signal amplification in a universal manner allowing faster rates of detection. We created Subzymes by manufacturing composite oligonucleotides that contain a catalytic nucleic acid component and a substrate component. We demonstrate that the activity of some catalytic DNAzyme components can be inhibited by attaching the Subzymes to micro-particles. Subsequent cleavage of a Subzyme's internal substrate results in the release and activation of the surface-bound DNAzyme, thus providing a mechanism to control the catalytic activity of the DNAzyme. We demonstrate that released DNAzymes are capable of cleaving fluorescent-labelled reporter substrates to generate a signal, thus confirming the restoration of their catalytic activity. The addition of Subzymes to reactions where the detection of a target is achieved by multi-component nucleic acid enzymes, known as PlexZymes, showed improved target sensitivities in a shorter amount of time (10 pM of target detected in under 60 min), demonstrating rapid detection of nucleic acid targets without the use of protein-enzymes. Subzymes are constructed from low-cost materials and operate under isothermal reaction conditions which are advantageous for on-site diagnostic testing. Thus, Subzymes offer a universal, rapid and affordable tool for nucleic acid sensing, providing new avenues for POC testing.

BACKGROUND:Whilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered.METHOD:PlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene.RESULTS:The combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%.CONCLUSION:PlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information.

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format.

Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.

We have created molecular switches that consist of nucleic-acid cleaving DNAzymes which are temporarily inactivated by hybridization with blocking oligonucleotides. The unique design of the switches offers significant advantages over existing methods. Firstly, the switches are activated by a nucleic acid-cleaving enzyme which can be made to function only in the presence of a specific target analyte. This allows for their use as reporter elements which can be easily adapted for use in computational logical operations. Secondly, the activation of each switch produces an active nucleic acid-cleaving DNAzyme as an output and this allows the switches to be modularly coupled to one another so that the output of one switch functions as the input of another. In addition, the switches are scalable, so that a single input target can produce more than one active DNAzyme output. These features therefore create the means for amplification of signal, which confers significant potential for future biosensing applications where detection of low quantities of target biomarkers is required.

Abstract Mutations in RAS, RAF, EGFR and PIK3CA genes are present in a number of cancers including colon, lung and breast cancers. Importantly, many of these mutations are predictive biomarkers of clinical outcomes for targeted therapies. This has led to a growing demand for detection of multiple mutations simultaneously. Multiplex formats allow a greater amount of information to be obtained from each sample. PASS primer technology can sensitively and specifically identify the presence of SNPs and somatic DNA changes (point mutations, deletions and insertions) with sensitivity up to 1 in 1,000 (0.1%). PASS primers selectively amplify nucleic acid variants and create amplicons that are markedly different from the parent sequence. The method is superior to ARMS PCR for multiplexing, particularly when mutations are present at the same, adjacent or nearby loci. PASS primers consist of two target specific regions separated by an insert sequence (IS) which is not complementary to the target. A long 5′ target-specific region anchors the primer and a short 3′ region targets the variant base/s thus directing highly specific binding and extension. During amplification, the IS sequences are incorporated into amplicons, introducing ≥10 base difference between the starting single base variant and the resulting amplicon. This reduces competition between primers during amplification and facilitates robust discrimination between amplicons during detection. MNAzyme® qPCR is a robust alternative to other probe based real-time qPCR protocols such as TaqMan® and Beacons. MNAzymes are bi-specific, catalytic oligonucleotide complexes which form in the present of target and cleave universal reporter probes. The bi-specificity, and the use of well-characterised universal probes sets (suitable for use with any group of targets), makes MNAzyme® qPCR more amenable to multiplexing. When combined with PASS, MNAzyme® qPCR easily discriminates between amplicons since MNAzymes are tailored to specifically detect both the distinct IS and the original mutation. PASS MNAzyme® qPCR has been used to facilitate multiplex detection of RAS, RAF, EGFR and PIK3CA mutations. As many as eight mutations located at codons 12 and 13 of RAS have been successfully detected in a single well using three channels on a standard PCR machine. The technology is even more powerful when combined with newer instruments with higher multiplex capabilities such as Biocartis’ Idylla automated platform. A multiplexed KRAS/BRAF assay, which detects 18 mutations, was evaluated using colon cancer and melanoma FFPE samples. The results showed >96% concordance with sequencing and significantly specificity compared to the singleplex Therascreen KRAS test. Since PASS MNAzyme® qPCR affords greater multiplex capacity, along with high specificity and sensitivity, it provides a superior tool for ascertaining mutations from tumour tissues and is particularly well suited for use with liquid biopsies. Note: This abstract was not presented at the meeting. Citation Format: Lit Yeen Tan, Elisa Mokany, Samantha Walker, Tina Lonergan, Alison Todd. Sensitive, specific and highly multiplexed mutation detection for cancer management. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4910. doi:10.1158/1538-7445.AM2015-4910

Diagnostic tests performed in the field or at the site of patient care would benefit from using a combination of inexpensive, stable chemical reagents and simple instrumentation. Here, we have developed a universal DNA-only Cascade (DoC) to quantitatively detect target analytes with increased speed. The DoC utilizes quasi-circular structures consisting of temporarily inactivated deoxyribozymes (DNAzymes). The catalytic activity of the DNAzymes is restored in a universal manner in response to a broad range of environmental and biological targets. The present study demonstrates DNAzyme activation in the presence of metal ions (Pb2+), small molecules (deoxyadenosine triphosphate) and nucleic acids homologous to genes from Meningitis-causing bacteria. Furthermore, DoC efficiently discriminates nucleic acid targets differing by a single nucleotide. When detection of analytes is orchestrated by functional nucleic acids, the inclusion of DoC reagents substantially decreases time for detection and allows analyte quantification. The detection of nucleic acids using DoC was further characterized for its capability to be multiplexed and retain its functionality following long-term exposure to ambient temperatures and in a background of complex medium (human serum).

Novel methods were developed to isothermally detect target nucleic acids and initiate signal amplification cascades. The methods utilize target-specific MNAzymes to activate universal primer molecules. These primers can subsequently promote the autonomous synthesis of DNA-zymes capable of cleaving nucleic acid substrates and generating signal, which further creates the potential for circular feedback.

Abstract Introduction The MAPK/ERK pathway is a complex signaling cascade involved in many cancer types. KRAS and BRAF gene mutations are present in a number of cancers, including colon, lung and pancreas, and identification of mutations in these genes is of great importance in clinical diagnostics. Moreover, there is a growing demand for performing multiple tests simultaneously on a single sample and there is an increased need to provide these answers to oncologists in a short timeframe. Methods IdyllaTM is a fully integrated and automated molecular diagnostics platform (1) that combines speed and ease of use with high sensitivity and high multiplexing capabilities. Moreover, it overcomes the current time-consuming step of processing formalin-fixed paraffin-embedded tissue (FFPE) samples. After insertion of a single FFPE slice into the cartridge, the complete process of sample preparation, real-time PCR and reporting is fully automated and takes less than 2 hours. We present here a KRAS-BRAF mutations prototype assay that allows the sensitive detection of 13 KRAS mutations and 5 BRAF mutations in one single assay. The assay discriminates the individual mutations at codons 12, 13 and 61 of KRAS and codon 600 of BRAF using novel “Primer Assisted Sequence Switching” (PASS) primers along with “Multi-component Nucleic Acid enzyme” (MNAzyme) detection. These technologies confer advantages for multiplex mutation analysis; PASS primers selectively amplify the target sequences of interest resulting in enhanced specificity between wild type (WT) and mutant, and between mutants, and MNAzymes allow for efficient detection and discrimination of multiple mutations simultaneously. Results Several performance characteristics of the IdyllaTM KRAS-BRAF prototype assay were examined: specificity, cross-reactivity, sensitivity and performance on clinical samples. Specificity of mutant versus WT as well as cross-reactivity between individual mutations at each codon was evaluated. Results demonstrated excellent specificity and cross-reactivity for all individual targets, with delta Cq values of >7 between mutants and >12 between mutant and WT. Sensitivity was assessed using cell lines embedded in FFPE containing defined ratios of mutants and dilutions of these in FFPE WT background. The results indicated sensitivities of <1% of mutant allele. The performance of the IdyllaTM KRAS-BRAF prototype assay was evaluated on a set of colon cancer and melanoma FFPE samples characterized by Sequenom MassARRAY, Illumina MiSeq, or Biocartis IdyllaTM BRAF-only assay which demonstrated a >96 % concordance. Conclusion The new and fully integrated IdyllaTM KRAS-BRAF prototype assay combines extended multiplexing capabilities with excellent specificity, high sensitivity, ease of use, and short turnaround time for mutation analysis on FFPE samples. (1) For research use only Citation Format: Ina Vandenbroucke, Katrien Vermeiren, Elisa Mokany, Lit Yeen Tan, Nicole Lima, Samantha Walker, Geneviève Vandercruyssen, Bart Claes, Inky De Baere, Pascale Holemans, Evelien Rondelez, Alison Todd, Geert Maertens, Erwin Sablon. A rapid and fully automated multiplex assay for KRAS-BRAF mutations with high mutation sensitivity using novel selective amplification and detection technologies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2014-1502