Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

Abstract

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™ 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 readout 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.

A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses

“…3B), and its selective ability for detecting sequences of strains haplotyped for the subgroup B (HU_14036/06). The increase in background signal when using the PLP cocktail can be attributed to the cumulative backgrounds from individual PLPs, and this can be addressed by decreasing the concentration of PLPs during the ligation step 20 . Also a careful selection must be performed to remove PLPs that alter the overall background performance of the assay.…”

“…Influenza type B/Stockholm/5/2014 (Victoria) strain was grown in Madin-Darby canine kidney cells (MDCK). Supernatants were obtained from cell culture as described previously 20 . Details of viral strains used in this study are summarized in SI, ST4.…”

“…Padlock probes (PLPs) in combination with Rolling Circle Amplification (RCA), an isothermal nucleic acid amplification method, comprise a robust molecular technique that has been used for molecular detection and typing of different pathogens 16–20 . A PLP is a single-stranded linear DNA oligonucleotide that contains 15–20 nt long target complementary arms and 40–50 nt non-hybridizing backbone.…”

Digital Rolling Circle Amplification–Based Detection of Ebola and Other Tropical Viruses

“…For the current study, two different versions of MRE chips were used: a pressure-driven MRE version 25 and a newly developed pump-free version.…”

“…22e24 Toward increased assay sensitivity, a new pump-free version of microfluidic RCP enrichment (MRE), based on capillary flow, was developed for digital quantification without specialized instrumentation. 25,26 Finally, the multiplexing capabilities of RCA was demonstrated by extending the EBOV PLP panel to Zika and Dengue by introducing appropriate barcode sequences in the PLP sets. Thus, the current study demonstrates a simplified RCA assay design for the clinical detection of EBOV, together with the multiplexing of tropical virus panel, using a comprehensive MRE strategy for obtaining digital counts of the analyte in the sample.…”