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Single tube Multiplex Amplification in Real Time

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 24 pathogens in a clinical sample.

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 instruments, PathoFinder has developed the SmartFinder® technology.

PathoFinder's 2SMART assays all make use of the same workflow based on this SmartFinder® technology. After a gene-specific multiplex reverse transcription step, a signal amplification step follows using a single PCR primer pair for all targets. One primer is labelled with a fluorescent dye (FAM, in 2SMART scheme blue probe). Simultaneously, an antificial sequence is incorporated in the reaction product (in 2SMART scheme, red sequence). The detection of the FAM labelled reaction product is done by melting curve analysis on real-time PCR instruments. Detection probes complementary to the incorporated artificial sequences, labelled with different fluorochromes and varying in melting temperature, enable specific detection of the reaction products and thus of the corresponding pathogens.

An Internal Control (IC) is added at the start of the procedure and detected by its own specific probe. The IC monitors nucleic acid extraction and amplification and validates in this way negative sample results.

Amplification Controls (AC) are added in the signal amplification step to be able to discriminate between possible nucleic acid extraction and amplification failure.

The 2Smart assays return results within 2.5 hours after nucleic acid extraction for respiratory tract infections, gastroenteritis, meningitis and sexually transmitted diseases, including viruses, bacteria and parasites.

Features and Benefits
  • Syndromic approach
  • Detection of up to 24 pathogens in a single reaction
  • Highly multiplexed PCR with a sensitivity equal to monoplex PCR