title: Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV.
abstract: Eleven new human polyomaviruses have been recently discovered, yet for most of these viruses, little is known of their biology and clinical impact. Rolling circle amplification (RCA) is an ideal method for the amplification of the circular polyomavirus genome due to its high fidelity amplification of circular DNA. In this study, a modified RCA method was developed to selectively amplify a range of polyomavirus genomes. Initial evaluation showed a multiplexed temperature-graded reaction profile gave the best yield and sensitivity in amplifying BK polyomavirus in a background of human DNA, with up to 1 × 10(8)-fold increases in viral genomes from as little as 10 genome copies per reaction. Furthermore, the method proved to be more sensitive and provided a 200-fold greater yield than that of random hexamers based standard RCA. Application of the method to other novel human polyomaviruses showed successful amplification of TSPyV, HPyV6, HPyV7, and STLPyV from low-viral load positive clinical samples, with viral genome enrichment ranging from 1 × 10(8) up to 1 × 10(10). This directed RCA method can be applied to selectively amplify other low-copy polyomaviral genomes from a background of competing non-specific DNA, and is a useful tool in further research into the rapidly expanding Polyomaviridae family.
authors.affiliation: Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Australia.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Rockett
authors.affiliation: Department of Renal Medicine, Royal Melbourne Hospital, Melbourne, Australia.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Barraclough
authors.affiliation: Department of Renal Medicine, Princess Alexandra Hospital, Brisbane, Australia.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Isbel
authors.affiliation: Institute for Future Environments, Central Analytical Research Facility, Queensland University of Technology, Brisbane, Australia.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Dudley
authors.affiliation: Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Australia.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Nissen
authors.affiliation: Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Australia.
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
authors.affiliation: Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Australia. Electronic address: seweryn@uq.edu.au.
authors.forename: Seweryn
authors.initials: S
authors.lastname: Bialasiewicz
mesh_terms: D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011120:Polyomavirus / Q000235:genetics / Q000302:isolation & purification*; D027601:Polyomavirus Infections / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral
keywords: BKV; Clinical sample; Low copy; Polyomavirus; Rolling circle amplification; Specific