The detection of influenza A and B viruses in clinical specimens using a quartz crystal microbalance

Abstract

Current methods for the accurate diagnosis of influenza based on culture of the virus or PCR are highly sensitive and specific but require specialised laboratory facilities and highly trained personnel and, in the case of viral culture, can take up to 14 days to obtain a definitive result. In this study, a quartz crystal microbalance-based immunosensor (QCM) has been developed and its potential evaluated for the rapid and sensitive detection of both influenza A and B viruses in laboratory-cultured preparations and clinical samples. The effective limit for detection by QCM for stock preparations of both A/PR/8/34 and B/Lee/40 viruses was 1 x 10(4) pfu/mL, associated with observed frequency shifts of 30 (+/-5) and 37 (+/-6.5) Hz, respectively. Conjugation of 13 nm gold nanoparticles to the detecting antibody improved the mass sensitivity of the immunosensor, resulting in a 10-fold increase in sensitivity and a detection limit of 1 x 10(3) pfu/mL for both preparations, with resulting frequency shifts of 102 (+/-11) and 115 (+/-5) Hz, respectively. Detection of virus in nasal washes with this technique was achieved by overnight passage in MDCK cultures prior to analysis. A comparison of results obtained from 67 clinical samples using existing RT-PCR, shell vial, cell culture and ELISA methods showed that QCM techniques were comparable in sensitivity and specificity to cell culture methods.

Fig. 1

Schematic of the apparatus used for continuous flow detection of influenza using a quartz crystal microbalance.

Fig. 2

Time-dependent frequency changes at different virus concentrations. (A) Frequency changes of the immunosensor using A/PR/8/34 virus. (B) Frequency changes of the immunosensor using B/Lee/40 virus. (C) Nanoparticle-conjugated anti-influenza A monoclonal antibody (A/PR/8/34). (D) Nanoparticle-conjugated anti-influenza B monoclonal antibody (B/Lee/40). () 10⁸ pfu/mL, () 10⁷ pfu/mL, (–■–) 10⁶ pfu/mL, () 10⁵ pfu/mL, (–▴–) 10⁴ pfu/mL, () 10³ pfu/mL, and () negative.

Fig. 3

Relationship between influenza virus concentration and frequency shifts in the presence and absence of nanoparticles. (A) A/PR/8/34. (B) B/Lee/40. (●) Without nanoparticles and (○) with nanoparticles.

Fig. 4

Time-dependent frequency changes of clinical samples in cell culture medium. (A) Frequency changes of clinical samples (without nanoparticles). (B) Frequency changes of clinical samples using nanoparticle-conjugated influenza A monoclonal antibody. () 10⁹ copies/mL, (–■–) 10⁶ copies/mL, () 10⁵ copies/mL, (–▴–) 10⁴ copies/mL, () 10³ copies/mL, and () negative.