Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Abstract

BackgroundState of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.Methodology and Principal FindingsThe described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.Conclusions and SignificanceThe system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.

Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique

“…Developers of POC viral load technologies are addressing this challenge either by building in an internal or external plasma separation step or by utilizing a more targeted biomarker. The Alere Q NAT POC assay (Alere Technologies, Jena, Germany) detects HIV-specific RNA and utilizes a smallvolume whole-blood input cartridge (6,7). This technology has recently been evaluated for the diagnosis of HIV in infants under 18 months of age in primary health care clinics, with promising results (8).…”

Detection and differentiation of HIV-2 using the point-of-care Alere q HIV-1/2 Detect nucleic acid test

“…After loading the sample capillary with 25 µL of plasma, each cartridge was inserted into an Alere q Analyzer where RNA isolation, cDNA synthesis, PCR and signal detection occur. The Alere q Analyzer reported either “detected” or “not detected” results for HIV-1 Group M/N, HIV-1 Group O and HIV-2 (18). The tests described in this study were carried out using four Alere q Analyzers in the Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited UW Clinical Retrovirus laboratory.…”

“…The Alere q HIV-1/-2 Detect (Alere Detect) test is a rapid point-of-care (POC) nucleic acid test (NAT) providing several advantageous characteristics for HIV diagnosis: 1) the requirement for only 25 microliters plasma or whole blood per test; 2) test completion within one hour; 3) detection and differentiation of HIV-1 Group M/N, HIV-1 Group O and HIV-2 nucleic acid; and 4) the only currently available POC NAT able to detect HIV-2 (18). Two studies have demonstrated the feasibility of the Alere Detect test to diagnose HIV-1 infection for HIV-1-exposed infants in South Africa and Mozambique (19, 20).…”

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

“…Nucleic acids amplification tests are important tools enabling sensitive, specific, and rapid detection of genetictargets 5 . Of these techniques, real-time polymerase chain reaction (rtPCR) is the basis for a majority of molecular assays 6 .…”

“…Microarrays area preferred method for the detection of multiple targets and these tools are expected to become a significant part of clinical diagnostic testing in the future along with next generation nucleotide sequencing methods 9, 10 . A major limitation hampering the implementation of microarrays in clinical setups derives from complex workflow, requiring numerous steps from sample to results 5, 11 . Furthermore, PCR produces double-stranded DNA targets requiring denaturation or preferably digestion of the complementary strand prior to hybridization onto capture probes immobilized on a solid support 12, 13 .…”