BACKGROUND: Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS: We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants-A(H1N1), A(H3N2), B/Yamagata, and B/Victoria-and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa (TM) Flu A/B & RSV Direct. RESULTS: The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS: We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital read-out for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are Limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (similar to 8 nL capture chamber) coupled with a thin-film photodiode (200 x 200 mu m area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 mu L of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.

Nucleic acid amplification tests have revolutionized the biomedical field by offering high sensitivity and specificity. Polymerase chain reaction (PCR) is considered as the gold standard for nucleic acid amplification; however, it requires sophisticated instrumentation for temperature cycling and real-time detection which makes it expensive. Isothermal amplification technologies have been developed to overcome these drawbacks, such as rolling circle amplification (RCA). In this work, we use the RCA and combine it with isotachophoresis (ITP) to increase the sensitivity for fluorescent real-time detection of nucleic acid amplification. For this, we use a top-down approach by first developing a suitable buffer system that supports RCA and ITP, and subsequently show the focusing of differently-sized and concentrated RCA products. Next, we compare our ITP-RCA assay with a commercial instrument for real-time fluorescence monitoring and demonstrate higher sensitivity from our method. Finally, we aim to combine the ligation and amplification step into ITP to simplify the RCA assay into a one-step reaction. The presented combination of RCA with ITP opens up new opportunities by making nucleic acid detection faster and simpler with potential applications for molecular diagnostics of infectious diseases.

Antibody characterization has become essential for diagnosis and development of therapeutic solutions in autoimmune, cardiovascular and infectious diseases. The specificity, affinity and isotype are crucial information for antibody studies and can be obtained from screening plasma samples or populations of B cells. Current technologies are mainly focusing on the discovery of abundant immunoglobulins, namely IgG, and are based on bulk measurements. In this study, we present a digital screening platform utilizing rolling circle amplification (RCA) for the detection of antigen-specific antibody isotypes in solution or secreted from single cells. To validate this approach, the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) was used as model disease and anti-ADAMTS13 antibodies were the target molecules. Antibody-oligonucleotide conjugates (AOCs) were designed for the multiplexed detection of human antibody isotypes IgA, IgG and IgM. Then, ADAMTS13 fragments were coated on glass slides and subsequently, target antibodies identified by specific AOC binding and visualized via an RCA assay. First, we validated the method by characterizing the assay specificity and limit of detection (LoD). When seeding different isotypes, we confirmed the high specificity of the assay (> 90%) and detection of monoclonal anti-ADAMTS13 IgG down to 0.3 ng/mL. A dilution series of a plasma sample of iTTP patient confirmed the multiplexed detection of the three isotypes with higher sensitivity compared to ELISA. Finally, we performed single cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving, achieving a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.