Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR.
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.
Berthet
Nicolas
N
1Genotyping of Pathogens and Public Health Technological Platform, Institut Pasteur, Paris, France. nberthet@pasteur.fr
Reinhardt
Anita K
AK
Leclercq
India
I
van Ooyen
Sven
S
Batéjat
Christophe
C
Dickinson
Philip
P
Stamboliyska
Rayna
R
Old
Iain G
IG
Kong
Katherine A
KA
Dacheux
Laurent
L
Bourhy
Hervé
H
Kennedy
Giulia C
GC
Korfhage
Christian
C
Cole
Stewart T
ST
Manuguerra
Jean-Claude
JC
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
D017931:DNA Primers; D004269:DNA, Bacterial; D012367:RNA, Viral; D004259:DNA-Directed DNA Polymerase
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