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258 Sensitive and Rapid Methods for Screening HIV-1 Subtype in Multiple Genomic Regions
M. Hoelscher1, J. K. Carr2*, V. Watanaveeradej3, J. Hierholzer2, J. L. Sanchez4, A. Brown5, D. Birx6, and F. McCutchan2
1Univ. of Munich, Germany; 2Henry M. Jackson Fndn., Rockville, MD, USA; 3Royal Thai Army, Bangkok, Thailand; 4US Naval Med. Res. Ctr., Lima, Peru; 5Armed Forces Res. Inst. of Med. Sci., Bangkok, Thailand; and 6Walter Reed Army Inst. of Res., Rockville, MD, USA
Background: The genetic diversity of HIV-1 presents continuing challenges in monitoring the strains in circulation in potential vaccine trial sites. The heteroduplex mobility assay (HMA) is a widely used tool but only surveys 1 or 2 regions of the genome and is labor-intensive.
Methods: HIV-1 DNA from PBMC was used as the substrate for real-time PCR, using a new approach with fluorescent-labeled, subtype-specific probes. One system, designed for Asia, can detect subtypes B, C, and CRF_AE. Another, for South America, can detect subtypes B and F. A third, designed for East Africa, can distinguish subtypes A, C, and D. Each assay scans 4 or 5 regions of the genome.
Results: These assays have high through-put, high sensitivity, and no background. Over 1,000 runs on samples from multiple sites have defined the performance of the assays, with sensitivity and specificity value estimates exceeding 80 and 98%, respectively. The assays can be used to detect and characterize recombinants by the simultaneous detection of different subtypes in multiple regions of the genome. One hundred samples can be scanned in 4 regions by 1 technician using 1 instrument in 1 week. The current reagent cost is approximately $10/sample.
Conclusions: A new method for screening DNA for HIV-1 genetic subtype has been developed which is quick and accurate. Since it screens multiple genome regions it can detect recombinants more readily than other assays.
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