Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

Nakamura, Shota; Yang, Cheng-Song; Sakon, Naomi; Ueda, Mayo; Tougan, Takahiro; Yamashita, Akifumi; Goto, Naohisa; Takahashi, Kazuo; Yasunaga, Teruo; Ikuta, Kazuyoshi; Mizutani, Tetsuya; Okamoto, Yoshiko; Tagami, Michihira; Morita, Ryoji; Maeda, Norihiro; Kawai, Jun; Hayashizaki, Yoshihide; Nagai, Yoshiyuki; Horii, Toshihiro; Iida, Tetsuya; Nakaya, Takaaki

Semi-quantitative PCR was performed using 10-fold serial dilutions of the amplified cDNA as templates. Flu-specific PCR detected positive signals in all three nasopharyngeal aspirates ( Figure 1B) , and norovirus-specific primer sets detected the norovirus genome in four fecal samples, excluding #N1 sample ( Figure 1C ). The endpoint of detection in sample #N3 without 70 cycles of PCR amplification was E+03 ( Figure 1C , left panel), whereas that with PCR amplification was E+06 ( Figure 1C , right panel), suggesting that the viral cDNA was amplified almost 1,000 times by the 70 cycles of PCR. Together with the results that total RNA/cDNA were also amplified from several nanograms (data not shown) to ,10 mg by random RT-PCR, these results indicate that viral genomes can be amplified similar to other DNAs with the WTA kit. Because almost all of the amplified cDNA were within the 200-1,000 bp range ( Figure 1A ), the PCR products were directly used as templates for emulsion PCR in the GS FLX pyrosequencing.