Biomedical subjects

J Sanders Sevall

Publications and source records attributed to J Sanders Sevall.

3 recordsLinked to original sources

PubMed2004

Molecular-based methods for quantifying HIV viral load.

Viral load quantitation has become the major prognostic marker for disease prognosis and outcome of antiretroviral therapy in the treatment of HIV-infected individuals. The three major methodologies for viral load quantitation: the reverse transcriptase-polymerase chain reaction (RT-PCR; Amplicor HIV-1 Monitor Test, Roche Diagnostic Systems, Pleasanton, CA), the nucleic acid sequence-based amplification (NASBA; NucliSens HIV-1 QT Test, Organon Teknika, Bostel, The Netherlands); and a signal amplification methodology termed branchedchain DNA (bDNA) technique (Quantiplex HIV-1 RNA test, Bayer Diagnostics, Emeryville, CA) are briefly reviewed here.

Biomarkers↗

PubMed2000

Factor V Leiden genotyping using real-time fluorescent polymerase chain reaction.

A fluorogenic probe-based PCR assay (Taqman; Perkin Elmer corp/Applied Biosystems, Foster City, CA, USA) was used for the detection of Factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for Deep Vein Thrombosis. This assay allows for the direct detection of specific PCR products within minutes of completion of the PCR by monitoring the increase in fluorescence of a dye-labelled oligonucleotide probe. Two dye-labelled probes are used in this allelic discrimination assay, one probe for each allele in the two-allele system. Each probe consists of an oligonucleotide with a 5'-reporter dye and a 3'-quencher dye. Tet (6-carboxy-4,7,2',7'-tetrachloro-fluorescein) is covalently linked to the 5'-end of the probe for the detection of allele 2 (wild-type). Fam (6-carboxy-fluorescein) is covalently linked to the 5'-end of the probe for detection of allele 1 (mutant). Each of the reporters is quenched by Tamra (6-carboxy-N,N,N', N'-tetramethylrhodamine) attached via a linker arm located at the 3'-end of each probe. The two probes were complementary to a 24-base sequence at the factor V Leiden mutation site, but differing in the 5'-labelled reporter dye and the nucleotide opposite the mutation site (C vs T). Wild-type and factor V Leiden alleles were differentiated in highly purified DNA and crudely purified DNA specimens. The assay was successfully applied to genomic DNA from leukocytes isolated from whole blood. The factor V status of 120 patients as determined by this method was in complete concordance with a standard PCR-based assay and clearly discriminated between healthy wild-type (+/+), factor V Leiden homozygote (-/-) and heterozygote (+/-) carriers.

Factor V↗

PubMedDate not supplied

Multiplex PCR using real time DNA amplification for the rapid detection and quantitation of HTLV I or II.

A multiplex 'real-time' polymerase chain reaction (PCR) has been established as a general technique for the quantitation of proviral human T-lymphotrophic virus types 1 and 2 (HTLV-I/II). The technology utilizes fluorescence to measure amplification products from the tax gene of Human T-cell lymphotropic virus type 1 or the 5' long terminal repeat of Human T-cell lymphotropic virus type 2. The quantitative amplification of the standard was linear across four orders of magnitude with nearly identical amplification efficiencies for monoplex or the biplex format from 1.4 copes/assay (60 copies proviral DNA/0.5 micrograms human DNA) to 6000 copies/assay (240000 proviral copies/0.5 micrograms human DNA). The human beta-globin gene was used to normalize for human DNA input to determine the proviral DNA load. Three hundred fifty-six specimens received by Specialty Laboratories for HTLV I/II detection provided identical results in the detection of HTLV I/II proviral DNA. No additional positive specimens were identified with the biplex assay format. The coefficient of variation for the proviral DNA load was less than 30% for HTLV I or II quantitation (n=5). For spiked specimens, two groups of five separate 0.25 ml blood specimens (20 total) were spiked, respectively, with 0, 9.6, 48, 240 and 1200 copies of HTLV I or HTLV II DNA standards. The specimens were amplified with the HTLV I/II multiplex format. Twenty of twenty expected negative HTLV I or HTLV II specimens were negative (100% specificity) and 14/16 specimens spiked with 48 copies or more HTLV I were detected (87.5% sensitivity). Thirteen of sixteen HTLV II spiked specimens (>48 copies of HTLV II standard per 10 assays) were detected (81.2%). The real-time detection provides accurate and reliable results in a single amplification for both HTLV (I or II) targets with a more rapid turnaround time and a decrease in material required for results.

DNA, Viral↗