Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR
1 Genotyping of Pathogens and Public Health Technological Platform, Institut Pasteur, Paris, France
2 Oncogenic Virus Epidemiology and Pathophysiology Unit, Institut Pasteur, Paris, France
3 Laboratory for Urgent Response to Biological Threats (CIBU), Institut Pasteur, Paris, France
4 Université Paris 7 Denis Diderot, Paris, France
5 Qiagen, Hilden, Germany
6 Affymetrix, Santa Clara, CA, USA
7 European Office, Institut Pasteur, Paris, France
8 Lyssavirus dynamics and host adaptation Unit, Institut Pasteur, Paris, France
9 Bacterial Molecular Genetics Unit, Institut Pasteur, Paris, France
BMC Molecular Biology 2008, 9:77 doi:10.1186/1471-2199-9-77Published: 4 September 2008
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).
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 × 107 using WTA, which yielded quantities of amplified DNA as high as 1.2 μg/μl or 1010 target copies. The amplification factor varied between 109 and 106. We also demonstrated that co-amplification occurred when viral RNA was mixed with bacterial DNA.
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.