Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform.</p><p><b>METHODS</b>Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance.</p><p><b>RESULTS</b>The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus.</p><p><b>CONCLUSION</b>The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.</p>

Cloning and Nucleotide Sequence Analysis of Aerolysin Gene of Aeromonas hydrophila / 微生物学通报

The aerolysin genes (aerA) of BZ and NK isolates were cloned and sequenced. The sequence analysis showed that the partial aerA of BZ and NK isolates consisted of 1393 bp, encoding a protein of 464 amino acids. The similarity of nucleotide and amino acid sequences of aerA between BZ and NK isolates was 97.6% and 98.3% respectively. The nucleotide sequence of aerA of BZ strain exhibited 71.6% to 97.5% homology with other Aeromonas isolates, and the amino acid sequence exhibited 68.0% to 98.9% homology. The phylogenetic tree based on aerA nucleotide sequences from Aeromonas isolates was constructed with neighbor-joining method. It showed that there were three branches of aerolysin genes, and a close relation- ship among Aeromonas hydrophila isolates which were clustered into the same branch.