Real time PCR on slide: a new tool for quantification of minute amounts of DNA

Abstract
Detection, identification and quantification of micro-organisms represents one of the major challenges for our modern society. It concerns a wide variety of applications including bioprocess control, food technology, health care, environmental analysis and of course clinical diagnostics. In this regard, miniaturization of PCR protocols may offer many advantages including short assay time, high precision and sensitivity, high-throughput, low reagent consumption and portability. Though on-chip PCR is likely to become the ‘next-generation PCR’, translating microfluidics to biology labs is still limited by its cost and the expertise it requires. In this context, a new format system has been developed, which allows DNA extraction and real-time PCR amplification in 1-µl “reaction sites” on a slide. The system, called SicLive, enables to process 48 samples in parallel and offers the possibility to perform quantitative PCR amplifications within 2 hours, including DNA extraction. Results, obtained on different model systems, showed that reproducibility and dynamic range were comparable to those obtained in 20 µl with conventional real-time PCR. In terms of sensitivity, SicLive was able to amplify DNA from 1-5 copies of target as well as from single cells. By combining DNA extraction and amplification in a single reaction site, SicLive is particularly well adapted for quantifying precious samples containing very low amounts of genetic material, such as clinical or environmental samples. Finally, for single-cell applications, this slide format ensures an easy visual control, with standard microscopy, of both quality and quantity of the templates loaded.