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Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza
Felix Neumann1, Iván Hernández-Neuta1, Malin Grabbe2,3
1Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
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.
Insights
A new multiplexed assay using padlock probes offers accurate detection and subtyping of seasonal influenza strains. This innovative diagnostic tool demonstrates high specificity and potential for widespread use in identifying influenza A and B.
Area of Science:
- Molecular Diagnostics
- Virology
- Biotechnology
Background:
- Influenza poses a significant global health threat, causing millions of severe cases and hundreds of thousands of deaths annually.
- Current diagnostic methods necessitate improvement to mitigate influenza's morbidity and economic impact.
Purpose of the Study:
- To develop a novel multiplexed diagnostic assay for the simultaneous detection and subtyping of seasonal influenza.
- To integrate this assay with an inexpensive digital quantification method for improved influenza diagnostics.
Main Methods:
- Development of a multiplexed assay utilizing padlock probes and rolling circle amplification.
- Simultaneous targeting of all 8 genome segments from four circulating influenza variants (A/H1N1, A/H3N2, B/Yamagata, B/Victoria).
- Validation using characterized virus isolates, patient nasopharyngeal swabs, and comparison with a commercial assay.
Main Results:
- The assay demonstrated a low limit of detection (18 viral RNA copies).
- Achieved 100% analytical and clinical specificity for differential detection and subtyping.
- Showcased diagnostic sensitivity of 77.5% for influenza detection and up to 73% for subtyping.
Conclusions:
- A proof-of-concept padlock probe assay with digital readout for influenza A and B detection and subtyping has been successfully developed.
- The assay's high specificity, multiplexing capability, and digital quantification present it as a promising diagnostic tool for seasonal influenza.
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