Technology base - Diagnostics

Pathogen identification

Accurate and definitive detection and identification of pathogens is essential for correct diagnosis and treatment of infectious diseases. Detection of pathogens in blood samples is challenging due to the strict requirements on specificity, sensitivity and time. Traditional bacterial identification relies primarily on culture-based methodologies requiring 12 to 24 h for isolation and an additional 24 to 48 h for species identification. Recently, molecular techniques have proven beneficial for rapid bacterial identification, such as MALDI-TOF MS or PCR. Q-linea´s technology for pathogen detection is based on highly specific padlock probes and circle-to-circle isothermal nucleic acid amplification technology (C2CA). This technology allows for substantially higher degree of multiplexing capability, compared to PCR-based techniques, and suitable for random access type of analysis. Padlock probes have been shown to allow up to 20.000-plex measurements. The molecular technologies are based on a solid scientific foundation described in more than 200 scientific publications in Science, Nature Genetics, Nature Biotechnology, Nature Methods and PNAS among others. The enabling technologies are continuously developed by leading scientists at Uppsala and Stockholm Universities and have, to date, formed the basis for four commercial companies, including Q-linea.


Q-linea molecular engine

Using a combination of padlock probes and several rolling circle amplification (RCA) steps, specific and sensitive pathogen identification directly in human blood can be achieved. The high multiplexing level, enabled by the specificity of padlock probes, allow many different bacteria to be detected simultaneous without the problems of cross-reactivity seen with multiplexed PCR. Highly specific and selective padlock probes become circularized upon hybridization to a specific target DNA sequence, and amplified through RCA. The long reaction product from the first round of RCA is cleaved into single copies of the template sequence by adding a restriction enzyme and an oligonucleotide that hybridizes to the product, forming a duplex at the restriction site. With an excess of added oligonucleotides, the cleavage product can be denatured and re-annealed to form circular structures that are closed by ligation. These circles can then be amplified using a second and third round of RCA, a mechanism called circle-to-circle amplification(C2CA).