SmartFinder®: Multiplex PCR Technology Based on Melting Curve Analysis

SmartFinder

The SmartFinder® technology is PathoFinder’s proprietary state-of-the-art multiplex PCR technology for Real Time Molecular Diagnostics. It enables Single tube Multiplex Amplification in Real Time (SMART) of up to 22 infectious pathogens in a single clinical sample.

The SmartFinder® technology uses target specific probes, which, after a ligation step, are amplified and detected in Real Time. The pathogen identification is based on Melting Curve analysis in different detection channels (FAM, ROX and Cy5). This reaction is preceded by a target specific amplification to ensure high specificity and sensitivity.

SmartFinder

Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, PathoFinder has developed the SmartFinder® technology. For every SmartFinder® reaction, up to 12 targets can be identified by means of melting curve analysis.

After a gene-specific pre-amplification two unique probes hybridize specifically to the pre-amplified DNA of each pathogen present in the clinical specimen. Hybridized probes are joined by a ligation step and subsequently amplified using a single PCR primer pair of which one primer is labelled with a  fluorescent dye (FAM).

The detection of the amplified FAM labelled probes is by Melting Curve Analysis on Real Time PCR systems. Twelve detection  probes, either ROX or Cy5 labelled and varying in melting temperature,  enable specific detection of the amplified probe and the corresponding pathogen. The Internal Control (IC), which is added at the beginning of the procedure, is detected by a specific detection probe in an additional channel to validate a negative sample result.

Our next generation SmartFinder® FAST assays return results within 4 hours after nucleic acid extraction for both respiratory and gastro-enteritis related pathogens, including viruses, bacteria and parasites. The SmartFinder® technology is universally applicable and is used in the following products:

Publications

In the attachments below you will find several scientific publications about our technologies and products.

The original RespiFinder® publication (paper, JCM 2008)382.67