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.

We present 2 cases of Mycoplasma genitalium infection that were successfully treated with moxifloxacin despite the presence of quinolone resistance-associated mutations in these strains.

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.

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.

BACKGROUND MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. METHODS We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. RESULTS A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted single-target assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r(2) values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a background of 1000-10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. CONCLUSIONS MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper.

Adenosine deaminase(ADA), purine nucleoside phosphorylase (PNP), 5′ nucleotidase (5′NT), ecto-5′NT, hypoxanthine-guanine phosphoribosyltransferase(HGPRT), adenine phosphoribosyltransferase(APRT), adenosine kinase(AK), AMP deaminase (AMPD) and adenylate kinase(AdKin) activities were assayed in leukemic cells from bone marrow and/or peripheral blood of 43 newly diagnosed children with acute lymphoblastic leukemia(ALL). These enzyme activities have been investigated in relation to some immunological markers. ADA activity was higher in E-rosette positive leukemia(E+ ALL), while HGPRT, APRT, PNP, 5′NT, ecto-5′NT and AdKin activities were found to be lower in E+ ALL as compared to E− ALL. In common ALL (cALL) antigen positive leukemia, mean ADA activity was significantly lower as compared to cALL− leukemia, whereas PNP, 5′NT, ecto-5′NT and AdKin activities were significantly higher. cALL cells with cytoplasmic immunoglobulin M(IgM) heavy chains were found to have mean 5′NT activities twice as high as cALL cells lacking cytoplasmic IgM heavy chains. In two patients who had surface immunoglobulins on their cell membranes, low 5′NT activities were found. When measuring enzyme activities after 2–4 days of prednisone monotherapy, only mean ADA and HGPRT activities decreased in non-B, non-T ALL. These decreases were not significant in T-ALL patients. Mean enzyme activities in the leukemic cells of five patients with relapse were comparable to those in newly diagnosed patients, except for 5′NT, which was found to be within the activity range of control peripheral blood lymphocytes. It is concluded that ADA and AdKin activities are suitable as markers for E+ ALL and cALL+ leukemias respectively. 5′NT might help to distinguish between cALL cells having and lacking pre-B characteristics. Since 5′NT activity may also be decreased in B-ALL, it is not suitable as a T-ALL marker. Enzymes of purine metabolism in leukemic relapse need further investigation.

To increase the versatility and utility of nucleic acid enzymes, we developed multicomponent complexes, known as MNAzymes, which produce amplified "output" signals in response to specific "input" signals. Multiple oligonucleotide partzymes assemble into active MNAzymes only in the presence of an input assembly facilitator such as a target nucleic acid. Once formed, MNAzymes catalytically modify a generic substrate, generating an amplified output signal that heralds the presence of the target while leaving the target intact. We demonstrated several applications including sensitive, isothermal target detection; discrimination of polymorphisms; and highly specific monitoring of real-time polymerase chain reaction (PCR). Furthermore, we showed their capacity to function as molecular switches and to work in series to create a molecular cascade. The modular nature of MNAzymes, together with the separation of input and output functionalities, provides potential for their integration into diverse devices such as diagnostic biosensors, molecular computers, and/or nanoscale machines.