hiv drug resistance surveillance using pooled pyrosequencing使用池焦磷酸测序艾滋病病毒耐药性监测.pdfVIP

HIV Drug Resistance Surveillance Using Pooled Pyrosequencing 1 ´ 1 2 3 1 1 2,3 Hezhao Ji , Nathalie Masse , Shaun Tyler , Ben Liang , Yang Li , Harriet Merks , Morag Graham , Paul 1 1 Sandstrom , James Brooks * 1 National HIV and Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada, 2 Genomics Core Facility, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada, 3 Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada Abstract Background: Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals. ¨ Methodology/Principal Findings: The protease region from 96 treatment naıve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were co