title: A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies.
abstract: OBJECTIVE
  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
  
  
  METHODS
  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
  
  
  CONCLUSIONS
  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
  
  
  CONCLUSIONS
  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.
authors.affiliation: School of Life Sciences, Queensland University of Technology, Brisbane, Qld, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Perrott
authors.forename: G
authors.initials: G
authors.lastname: Smith
authors.forename: Z
authors.initials: Z
authors.lastname: Ristovski
authors.forename: R
authors.initials: R
authors.lastname: Harding
authors.forename: M
authors.initials: M
authors.lastname: Hargreaves
mesh_terms: D000336:Aerosols; D000391:Air Microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000336:Aerosols; D012367:RNA, Viral