Size exclusion-based purification and PCR-based quantitation of MS2 bacteriophage particles for environmental applications

摘要

MS2 bacteriophage is the most commonly used surrogate for pathogenic viruses in laboratory and field studies. In order to determine the number of infectious viral particles in samples, the use of accurate quantitation methods is essential. We have optimised a size exclusion chromatography-based method for MS2 purification and a SYBR Green-based single-step quantitative reverse transcriptase polymerase chain reaction (gRT-PCR) assay for the quantitation of MS2. The gRT-PCR enabled accurate quantitation of viral RNA of the purified stock with a detection limit of 2 genome copy equivalents/mu l. Detection inhibition, if any, was eliminated by reducing sample volume added to the gRT-PCR reaction mix when MS2 was detected in environmental water samples. The purification method eliminated the impurities and the purified stock yielded a high concentration of infectious MS2 particles. The gRT-PCR assay enabled the accurate,quantitation of the viral particles thus providing an alternative to the traditional plaque assays. A combined use of purified MS2 stock and PCR-based quantitation gives the opportunity to explore virus characteristics, behaviour and interactions in the environment. (C) 2014 Elsevier B.V. All rights reserved.