Subtyping clinical specimens of influenza A virus by use of a simple method to amplify RNA targets.

This work presents the clinical application of a robust and unique approach for RNA amplification, called a simple method for amplifying RNA targets (SMART), for the detection and identification of subtypes of H1N1 pandemic, H1N1 seasonal, and H3N2 seasonal influenza virus. While all the existing amplification techniques rely on the diffusion of two molecules to complex RNA structures, the SMART achieves fast and efficient amplification via single-molecule diffusion. The SMART utilizes amplifiable single-stranded DNA (ssDNA) probes, which serve as reporter molecules for capturing specific viral RNA (vRNA) sequences and are subsequently separated on a microfluidic chip under zero-flow conditions. The probe amplification and detection are performed using an isothermal (41°C) amplification scheme via a modified version of nucleic acid sequence-based amplification (NASBA). In our study, 116 consecutive, deidentified, clinical nasopharyngeal swab samples were analyzed independently in a blinded fashion using the SMART, reverse transcription-PCR (RT-PCR), antigen (Ag) testing, and viral culture. The SMART was shown to have a limit of detection (LOD) of approximately 10(5) vRNA copies/ml, corresponding with a time-to-positivity (TTP) value of 70 min for real-time detection. The SMART correctly detected influenza virus in 98.3% of the samples with a subtyping accuracy of 95.7%. This work demonstrates that the SMART represents a highly accurate diagnostic platform for the detection and subtyping of influenza virus in clinical specimens and offers significant advantages over the current commercially available diagnostic tools.