Real-time apta-PCR for 20 000-fold improvement in detection limit

Abstract

A real-time apta-PCR for the ultrasensitive detection of thrombin is reported, where the thrombin aptamer acts not only as a biomolecular recognition element, but also as a label for amplification via real-time PCR. Aptamers can be easily converted to a reporter agent for detection by real-time PCR, simply via flanking of the aptamer's recognition moiety with primer sequences. The reported technique has the advantage of the ultrasensitivity achievable with immuno-PCR, but without the complications of addition of a DNA label, and is a technique generically applicable to all aptamers. Here, we use a sandwich format, where two existing thrombin binding aptamers with distinct binding epitopes have been utilised to capture and detect thrombin in a streptavidin-coated microtiter plate. The amount of thrombin is calculated from real-time PCR analysis of eluted captured reporter aptamer. However, the technique can also be used for aptamer-antibody sandwiches, or simply with single aptamers. A greater than 20 000-fold increase in sensitivity is achieved, highlighting the potential of this approach for the detection of very low levels of target analytes. The use of the aptamer itself as the reporter molecule eliminates the necessity of laborious enzyme/DNA labelling, facilitating a significantly more straightforward assay with a vastly enhanced sensitivity.