Abstract: We present a highly specific, sensitive and cost-effective system to quantify miRNA, for typing of cell-free DNA in liquid biopsies and for direct blood genotyping based on novel chemistry called Two-tailed PCR. Two-tailed PCR takes advantage of target-specific primers composed of two hemiprobes complementary to two different parts of the target molecule connected by a hairpin structure. The introduction of a short hemiprobe that senses the variable sequences confers exceeding sequence specificity while maintaining the very high sensitivity of PCR. Highly similar targets can be distinguished with superior precision irrespectively of the position of the mismatched nucleotide. Further, the target molecule can be very short, making Two-tailed PCR the preferred method for microRNA profiling as well as analysis of rare sequence variants in cell-free (cf) DNA and formalin fixed paraffine embedded (FFPE) tissue. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity sufficient to detect less than ten target miRNA molecules. Two-tailed PCR is readily multiplexed and for less challenging applications such as genotyping Two-tailed PCR can be applied directly on blood samples eliminating the need for extraction. This very smooth and friendly workflow is most suitable point-of-care testing at bedside, doctor’s office or in the field. Applications for cfDNA analysis are being developed by SimSen Diagnostics AB.

(1) Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. P Androvic, L Valihrach, J Elling, R Sjoback, M Kubista. Nucleic acids research 45 (15), e144-e144

Biography: With a background as professor in Chemistry Kubista has become a serial entrepreneur in diagnostics. In 1996 he invented the light-up probes, which led to the foundation of LightUp Technologies AB as Europe´s first company focusing on qPCR-based diagnostics. In 2001 Kubista set up TATAA Biocenter as the first laboratory in Europe to obtain flexible ISO 17025 accreditation focuses on advanced services in molecular analyses to the pharmaceutical and biotech industries. in 2019 TATAA Biocenter was named “Best Nucleic Acid Analysis Service Provider – Europe” by Global Health & Pharma. Kubista introduced non-invasive prenatal testing (NIPT) in Sweden founding Life Genomics AB and direct blood genotyping co-founding LifeTest. Most recently TATAA invented the Two-tailed PCR for ultrasensitive nucleic acid detection, which has been licensed to Biovendor for microRNA analysis, and most recently SimSen Diagnostics was co-founded with the inventors of SimSen Sequencing to developed an ultrasensitive methods for molecular analyses of liquid biopsies. During the pandemic TATAA Biocenter was among the first in Europe testing for SARS-CoV-2 corona virus starting February 1, and is now contributing to the Swedish public testing effort as well as organizing testing of travellers through the platform www.coronapassport.se.