Outbreak of febrile respiratory illness associated with human adenovirus type 14p1 in Gansu Province, China

Huang, Guohong; Yu, Deshan; Zhu, Zhen; Zhao, Hai; Wang, Peng; Gray, Gregory C.; Meng, Lei; Xu, Wenbo

Viral DNA was extracted from HAdV isolates by using a QIAamp DNA mini kit (Qiagen, Valencia, CA, USA). HAdV type was identified by amplifying and sequencing the complete HAdV hexon, fiber, and E1A genes. Gene amplification was performed using a GeneAmp 9700 thermal cycler (Applied Biosystems, Carlsbad, CA, USA). Briefly, the PCR chemistry included a 25-ll reaction mixture containing 2 9 PCR Mix (Promega, Fitchburg, WI, USA), 1 ll of each primer (listed in Table 1 ), and 2 ll of template DNA. PCR conditions included an initial denaturation at 94°C for 10 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 70 seconds, and a final extension at 72°C for 10 minutes. The amplification products were prepared through capillary gel electrophoresis using the QIAxcel DNA High Resolution Kit (Qiagen, Venlo, the Netherlands). The PCR products were purified (Qiagen, Valencia, CA, USA) and sequenced using the dye terminator method (BigDye Terminator, version 3.1, cycle sequencing kit; Applied Biosystems) with an ABI Prism 3100 genetic analyzer (Applied Biosystems).