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J Clin Microbiol. 2013 Jul 31. [Epub ahead of print]
Subtyping Clinical Specimens of Influenza A Virus using a Simple Method to Amplify RNA Targets (SMART).
Wang J, Tai W, Angione S, John AR, Opal SM, Artenstein AW, Tripathi A.
Source
Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI 02912.
Abstract
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 subtype identification 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 method achieves fast and efficient amplification via single molecule diffusion. 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 is performed using an isothermal (41?C) amplification scheme via a modified version of nucleic acid sequence based amplification (NASBA). In our study, 116 consecutive, de-identified, clinical nasopharyngeal swab samples were analyzed independently and in a blinded fashion using SMART, RT-PCR, antigen (Ag) testing, and by viral culture. SMART was shown to have a limit of detection (LOD) of approximately 105 vRNA copies/mL, corresponding with a time-to-positivity (TTP) value of 70 minutes for real-time detection. SMART correctly detected influenza in 98.3% of the samples with a subtyping accuracy of 95.7%. This work demonstrates that SMART represents a highly accurate diagnostic platform for the detection and subtyping of influenza virus in clinical specimens and offers significant advantages over current commercially available diagnostic tools.
PMID:
23903546
[PubMed - as supplied by publisher]
Subtyping Clinical Specimens of Influenza A Virus using a Simple Method to Amplify RNA Targets (SMART).
Wang J, Tai W, Angione S, John AR, Opal SM, Artenstein AW, Tripathi A.
Source
Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI 02912.
Abstract
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 subtype identification 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 method achieves fast and efficient amplification via single molecule diffusion. 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 is performed using an isothermal (41?C) amplification scheme via a modified version of nucleic acid sequence based amplification (NASBA). In our study, 116 consecutive, de-identified, clinical nasopharyngeal swab samples were analyzed independently and in a blinded fashion using SMART, RT-PCR, antigen (Ag) testing, and by viral culture. SMART was shown to have a limit of detection (LOD) of approximately 105 vRNA copies/mL, corresponding with a time-to-positivity (TTP) value of 70 minutes for real-time detection. SMART correctly detected influenza in 98.3% of the samples with a subtyping accuracy of 95.7%. This work demonstrates that SMART represents a highly accurate diagnostic platform for the detection and subtyping of influenza virus in clinical specimens and offers significant advantages over current commercially available diagnostic tools.
PMID:
23903546
[PubMed - as supplied by publisher]
