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Clin Chem. 2018 Sep 26. pii: clinchem.2018.292979. doi: 10.1373/clinchem.2018.292979. [Epub ahead of print]
Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza.
Neumann F1, Hern?ndez-Neuta I1, Grabbe M2,3, Madaboosi N1, Albert J2,3, Nilsson M4.
Author information
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.
? 2018 American Association for Clinical Chemistry.
PMID: 30257827 DOI: 10.1373/clinchem.2018.292979
Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza.
Neumann F1, Hern?ndez-Neuta I1, Grabbe M2,3, Madaboosi N1, Albert J2,3, Nilsson M4.
Author information
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.
? 2018 American Association for Clinical Chemistry.
PMID: 30257827 DOI: 10.1373/clinchem.2018.292979