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Virus type-specific DNA-PCR detection of HIV-1 group M, group O, and HIV-2 isolates using an efficient 96-well plate format.
Fridlund C; Otten RA; Adams DR; Downing RG; Anyaegani G; Hu D; Wright
July 30, 1997
4th Conf Retro and Opportun Infect. 1997 Jan 22-26;:116 (abstract no.

A network of field sites exists both in the United States and in collaborating centers abroad (Africa, Asia, Latin America, and the Caribbean) in which the prevalence, distribution, and global transmission of diverse HIV strains is being monitored. A major goal of these efforts is a rigorous attempt to ensure the effectiveness of HIV diagnostic tests in protecting both international and domestic blood supplies. In many of these sites such as Uganda, serologic surveys for different HIV subtypes have been hampered by broad cross-reactivity to peptides encompassing virus subtype-specific, immunogenic regions. As a result, highly detailed virologic studies at the genetic level have to be carried out in order to gain the knowledge desired. To this end, we have exploited and adapted a commercially available system for use in molecularly typing distinct viruses. Creation of cellular lysates on site greatly facilitated genetic investigations of proviral DNA using PCR. Primers and oligonucleotide probes specific for HIV-1 group M, group O, or HIV-2 strains have been developed using the conserved protease gene located in the viral pol region. The PCR ELISA format provides for specific detection of PCR products in a semi-quantitative manner. Digoxigenin-dUTP is utilized to label amplicons generated by PCR. Exclusive hybridization of a virus type-specific, biotinylated probe to DIG-labeled amplicons and subsequent capture on a streptavidin coated microtiter plate surface are then performed. Bound hybrids are detected by the reaction of an anti-digoxigenin peroxidase conjugate with the appropriate colorimetric substrate. This ongoing project should provide for a more complete understanding of the extent of evolution of these viruses and their potential threat to blood supplies in the future.