Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

Baldwin, Don A.; Feldman, Michael; Alwine, James C.; Robertson, Erle S.

Three whole-genome amplification (WGA) methods that use phi29 polymerase rolling-circle amplification (24) (GenomiPhi), universal primer multiplex PCR (25) (GenomePlex and Trans-Plex), or single-primer isothermal amplification (26) (Ovation WGA) were tested for their ability to detect a small bacteriophage genome spiked into a background of human DNA (15 ng). phiX174 DNA at copy numbers 1Ï«, 10Ï«, and 100Ï« relative to a single-copy human genomic locus was easily detected by PathoChip probes after any of the amplification reactions ( Fig. 2A) . To test detection of human DNA and RNA viruses, DNA from cell lines containing adenovirus type 5 or RNA containing respiratory syncytial virus was amplified by the Genome-Plex DNA and TransPlex RNA methods (experiments 2 and 3, Table 2 ). The cell line DNA and RNA samples were then mixed and simultaneously amplified by TransPlex (experiment 4, Table 2 ). Probes for both viruses produced strong and specific Baldwin et al. detection signals. This indicated that the TransPlex reverse transcription worked robustly in the presence of genomic DNA, and genomic DNA and cDNA targets were coamplified.