title: Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR.
abstract: BACKGROUND
  Phi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments (<2000 bases) obtained by reverse transcription of certain viral RNA genomes. Therefore, ligation of cDNA fragments is necessary prior phi29 polymerase based amplification. We adapted the QuantiTect Whole Transcriptome Kit (Qiagen) to our purposes and designated the method as Whole Transcriptome Amplification (WTA).
  
  
  RESULTS
  WTA successfully amplified cDNA from a panel of RNA viruses representing the diversity of ribovirus genome sizes. We amplified a range of genome copy numbers from 15 to 4 x 10(7) using WTA, which yielded quantities of amplified DNA as high as 1.2 microg/microl or 10(10) target copies. The amplification factor varied between 10(9) and 10(6). We also demonstrated that co-amplification occurred when viral RNA was mixed with bacterial DNA.
  
  
  CONCLUSIONS
  This is the first report in the scientific literature showing that a modified WGA (WTA) approach can be successfully applied to viral genomic RNA of all sizes. Amplifying viral RNA by WTA provides considerably better sensitivity and accuracy of detection compared to random RT-PCR.
authors.affiliation: 1Genotyping of Pathogens and Public Health Technological Platform, Institut Pasteur, Paris, France. nberthet@pasteur.fr
authors.forename: Nicolas
authors.initials: N
authors.lastname: Berthet
authors.forename: Anita K
authors.initials: AK
authors.lastname: Reinhardt
authors.forename: India
authors.initials: I
authors.lastname: Leclercq
authors.forename: Sven
authors.initials: S
authors.lastname: van Ooyen
authors.forename: Christophe
authors.initials: C
authors.lastname: Batéjat
authors.forename: Philip
authors.initials: P
authors.lastname: Dickinson
authors.forename: Rayna
authors.initials: R
authors.lastname: Stamboliyska
authors.forename: Iain G
authors.initials: IG
authors.lastname: Old
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Kong
authors.forename: Laurent
authors.initials: L
authors.lastname: Dacheux
authors.forename: Hervé
authors.initials: H
authors.lastname: Bourhy
authors.forename: Giulia C
authors.initials: GC
authors.lastname: Kennedy
authors.forename: Christian
authors.initials: C
authors.lastname: Korfhage
authors.forename: Stewart T
authors.initials: ST
authors.lastname: Cole
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Manuguerra
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D020869:Gene Expression Profiling / Q000379:methods*; D016679:Genome, Viral; D023281:Genomics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012328:RNA Viruses / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012296:Rift Valley fever virus / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012367:RNA, Viral; D004259:DNA-Directed DNA Polymerase
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