Rapid subtype-specific detection of respiratory syncytial virus A and B using an RNase HII-dependent RT-LAMP lateral flow assay
Abstract
Introduction Rapid and simultaneous detection of respiratory syncytial virus A (RSV A) and RSV B with subtype discrimination is important for point-of-care (POC) diagnostics and epidemiological surveillance. Methods We developed the RSV A/B rh-RT-LAMP-NALF assay, a duplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) system combined with nucleic acid lateral flow (NALF) detection for simultaneous subtype-specific detection of RSV A and RSV B from nasopharyngeal swab specimens. To improve specificity during multiplex RT-LAMP, blocked inner primers, activated by RNase HII, were incorporated to suppress non-specific amplification. Subtype-specific detection was achieved on a single PCRD multiplex strip using 5′-FAM–and 5′-DIG–labeled loop primers for RSV A (T1 line) and RSV B (T2 line), respectively. Results The analytical limit of detection was 4 × 101 copies/μL using plasmid DNA standards. In clinical specimen dilution series, the assay detected RSV down to the 10−3 dilution in clinical specimens, showing sensitivity approaching that of the comparator RT-PCR assay. Clinical validation was performed using 281 archived nasopharyngeal swab specimens (87 RSV A-positive, 94 RSV B-positive, and 100 RSV-negative), with the Allplex™ Respiratory Panel 1 assay used as the reference method. The RSV A/B rh-RT-LAMP-NALF assay demonstrated sensitivities of 95.4% (95% CI: 88.8%–98.2%) for RSV A and 97.9% (95% CI: 92.6%–99.4%) for RSV B, with no false-positive results observed in the 100 RSV-negative specimens. These results were comparable to those of the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (RSV A: 95.4%; RSV B: 98.9%) and higher than those of the STANDARD Q RSV Ag Test (RSV A: 82.8%; RSV B: 92.6%). Limited cross-subtype reactivity was observed in one RSV A-positive specimen and one RSV B-positive specimen, both of which showed dual T1/T2 signals on the lateral flow strip. No cross-reactive signals were observed in 42 specimens representing 14 common respiratory pathogens other than RSV. Discussion Overall, the RSV A/B rh-RT-LAMP-NALF assay provides a rapid, accurate, and subtype-specific POC diagnostic platform for RSV detection and surveillance.