title: Size exclusion-based purification and PCR-based quantitation of MS2 bacteriophage particles for environmental applications.
abstract: 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 (qRT-PCR) assay for the quantitation of MS2. The qRT-PCR enabled accurate quantitation of viral RNA of the purified stock with a detection limit of 2 genome copy equivalents/μl. Detection inhibition, if any, was eliminated by reducing sample volume added to the qRT-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 qRT-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.
authors.affiliation: Institute of Environmental Science & Research Ltd, PO Box 29181, Christchurch 8540, New Zealand; School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand. Electronic address: fkata211@gmail.com.
authors.forename: Kata
authors.initials: K
authors.lastname: Farkas
authors.affiliation: School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand; Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa.
authors.forename: Arvind
authors.initials: A
authors.lastname: Varsani
authors.affiliation: School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand.
authors.forename: Delphine
authors.initials: D
authors.lastname: Marjoshi
authors.affiliation: Otago Centre for Electron Microscopy, University of Otago, PO Box 56, Dunedin, New Zealand.
authors.forename: Richard
authors.initials: R
authors.lastname: Easingwood
authors.affiliation: Institute of Environmental Science & Research Ltd, PO Box 29181, Christchurch 8540, New Zealand.
authors.forename: Erin
authors.initials: E
authors.lastname: McGill
authors.affiliation: Institute of Environmental Science & Research Ltd, PO Box 29181, Christchurch 8540, New Zealand.
authors.forename: Liping
authors.initials: L
authors.lastname: Pang
mesh_terms: D002850:Chromatography, Gel / Q000379:methods*; D004783:Environmental Microbiology; D017909:Levivirus / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019562:Viral Load / Q000379:methods*
keywords: MS2 bacteriophage; Size exclusion chromatography; Virus purification; qRT-PCR