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

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

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Molecular BioSystems Vol - 5 Issue - 4

Molecular BioSystems Vol - 5 Issue - 3

Molecular BioSystems Vol - 5 Issue - 2

Molecular BioSystems Vol -4

Molecular BioSystems Vol -3

Molecular BioSystems Vol -2

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

Content Provider	Royal Society of Chemistry (RSC)
Author	Pinto, Alessandro Redondo, M. Carmen Bermudo O’Sullivan, Ciara K. Ozalp, V. Cengiz
Copyright Year	2009
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.
Starting Page	548
Ending Page	553
Page Count	6
File Format	HTM / HTML PDF
ISSN	1742206X
Volume Number	5
Issue Number	5
Journal	Molecular BioSystems
DOI	10.1039/b814398f
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Polymerase chain reaction Reporter gene Microtiter plate Enzyme DNA Thrombin Aptamer
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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