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Development of a quantitative PCR assay for HIV-l using a bioluminescent detection system in an ELISA-plate format.
Guenthner PC; Hart CE; Centers for Disease Control and Prevention,
November 30, 1996
3rd Conf Retro and Opportun Infect. 1996 Jan 28-Feb 1;:155. Unique

We have developed a quantitative PCR assay for detecting human immunodeficiency virus type-1 in a broad range of source materials. The quantitative PCR is a modification of the technique described by Piatek that uses a cloned fragment or transcript (for DNA or RNA PCR) of the HIV-1 gag gene that contains a deletion as a competitive internal standard during PCR We modified this internal standard by incorporating a 31bp insert from pBR327 as a site for probe hybridization to eliminate crossreactivity between probes. PCR is performed using primers to HIV-1 gag with a 5' biotinylated primer. To a constant amount of test sample, 10(4) to 10 copies in 10-fold dilutions of the internal standard are added. Aliquots of the PCR reactions are hybridized to a streptavadin-coated microtiter plate and denatured by alkali. The resulting bound single-stranded PCR products are hybridized to either a wild-type or internal standard HIV-1 probe labeled with digoxigenin (DIG), followed by an c-x-DIG antibody conjugated to the bioluminescent protein aequorin (SeaLite Sciences). The aequorin is then detected by flash luminometry. This assay is linear over the range of 10(4) to 10 copies, and consists of a simple set of PCR or RT-PCR reactions and an easily manipulated ELISA-plate format. The standards are subjected to all the same conditions as the test samples. This assay can be carried out on materials from a broad range of sources, such as plasma tissue extractions, cultured cells, and genomic DNA. Current tests of the assay include quantitation of viral RNA in plasma and in cervical/vaginal lavage, and of proviral DNA in paraffin tissue sections. The assay was not inhibited by any of these source materials. 1 BioTechniques( 1993)14:70-81.

DNA Primers DNA Probes DNA, Viral/BLOOD Enzyme-Linked Immunosorbent Assay Genes, gag HIV-1/GENETICS/*ISOLATION & PURIF Human Luminescence Nucleic Acid Denaturation Nucleic Acid Hybridization Polymerase Chain Reaction RNA, Viral/BLOOD Reference Standards ABSTRACT