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A nested real-time PCR assay has an increased sensitivity\ud suitable for detection of viruses in aerosol studies

By Phillipa Perrott, Greg A. Smith, Zoran Ristovski, Robert M. Harding and Megan Hargreaves

Abstract

Aims: Influenza is commonly spread by infectious aerosols; however, detection\ud of viruses in aerosols is not sensitive enough to confirm the characteristics of\ud virus aerosols. The aim of this study was to develop an assay for respiratory\ud viruses sufficiently sensitive to be used in epidemiological studies.\ud Method: A two-step, nested real-time PCR assay was developed for MS2 bacteriophage,\ud and for influenza A and B, parainfluenza 1 and human respiratory\ud syncytial virus. Outer primer pairs were designed to nest each existing real-time\ud PCR assay. The sensitivities of the nested real-time PCR assays were compared\ud to those of existing real-time PCR assays. Both assays were applied in an aerosol\ud study to compare their detection limits in air samples.\ud Conclusions: The nested real-time PCR assays were found to be several logs\ud more sensitive than the real-time PCR assays, with lower levels of virus\ud detected at lower Ct values. The nested real-time PCR assay successfully\ud detected MS2 in air samples, whereas the real-time assay did not.\ud Significance and Impact of the Study: The sensitive assays for respiratory\ud viruses will permit further research using air samples from naturally generated\ud virus aerosols. This will inform current knowledge regarding the risks associated\ud with the spread of viruses through aerosol transmission

Topics: 060506 Virology, aerosolization, bacteriophage, PCR, respiratory virus
Publisher: Blackwell Publishing
Year: 2009
DOI identifier: 10.1111/j.1365-2672.2008.04119.x
OAI identifier: oai:eprints.qut.edu.au:29270

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