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.

Highlights

  • Simultaneous sensitive detection and quantification of multiple molecular targets are key prerequisites for the development of state of the art molecular diagnostic tests [1,2,3,4,5]

  • 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

  • High sensitivity is required for most diagnostic applications, in the monitoring of blood borne pathogens like human immunodeficiency virus (HIV) or hepatitis-C virus (HCV) [6,7]

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Summary

Introduction

Simultaneous sensitive detection and quantification of multiple molecular targets are key prerequisites for the development of state of the art molecular diagnostic tests [1,2,3,4,5]. One particular advantage of real time PCR assays is that they can simultaneously amplify and detect targets. One inherent limitation of assays based on probe array readout is that their workflows require numerous steps to be performed. For target quantification a linear or competitive amplification format is usually used, which substantially limits the sensitivity of the assays. Amplification, probe-array hybridization, and detection are usually separate steps that require specific reagents and conditions. State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. 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

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