Biomedical subjects

J Morlese

Publications and source records attributed to J Morlese.

3 recordsLinked to original sources

PubMed2002

Reliable and reproducible LightCycler qPCR for HIV-1 DNA 2-LTR circles.

Highly active anti-retroviral therapy (HAART) has reduced the plasma load of HIV-1 to undetectable levels. It has however failed to eliminate the virus from other body compartments. Current methods for monitoring persistent viral replication in HIV-1+ patients require a large amount of blood and/or repeated tissue biopsies. Furthermore, some of the viral reservoirs, such as brain and eye, are inaccessible for sampling. The detection of episomal HIV-1 DNA 2-LTR circles in CD4+ cells is indicative of recent, acute infection events. This paper describes a reliable and reproducible LightCycler-based assay for the quantitative measurement of HIV-1 DNA 2-LTR circles in human peripheral blood mononuclear (PBMN) cells. It details the modifications to the DNA extraction procedure and to the LightCycler PCR procedure that were required to achieve this. This new surrogate marker of persistent viral replication can now be reliably, reproducibly and robustly used to study the clinical progress of large numbers of patients whose plasma HIV-1 RNA has been reduced to undetectable levels by anti-retroviral drugs.

DNA Restriction Enzymes↗

PubMed2002

LightCycler qPCR optimisation for low copy number target DNA.

The LightCycler is a rapid air-heated thermal cycler which incorporates a fluorimeter for the detection and quantification of Polymerase Chain Reaction (PCR) amplified products. It provides real-time cycle-by-cycle analysis of product generation. Amplification occurs in glass capillary tubes. The products are detected using a fluorescent double stranded DNA binding dye or fluorescent probes. However, conditions that work well in conventional PCR reactions do not readily translate to the LightCycler. Whilst using this new technology to study an infectious pathogen in human tissue samples, several parameters were identified which can have an adverse effect on the reliable and reproducible quantification of low copy number target DNA. They included abstraction of PCR reagents on glass, primer-dimer formation, non-specific product generation, and a failure to amplify low copy number target when it is present in a high background of human chromosomal DNA. For each problem identified, several solutions are described. Novel approaches are also described to ensure that amplification of target DNA and of the quantification standards occurs with the same efficiency. With appropriate changes to the protocols currently in use, LightCycler quantitative Polymerase Chain Reaction (LC-qPCR) can be used to achieve a level of accuracy that exceeds that of an enzyme immunoassay. The LC-qPCR optimisation strategies described are of particular relevance when applying this technology to the study of pathogens in tissue samples. The technique offers the enormous potential for reliable and reproducible quantitative PCR of low copy number target DNA.

DNA Primers↗

PubMed2000

Diagnosis and management of male breast enlargement in patients with HIV/AIDS.

There have been several recent reports describing gynecomastia in HIV-1-seropositive patients treated with HAART. However, the etiology of gynecomastia in this setting is far from clear. In this article, we describe the main issues in diagnosis and treatment of gynecomastia and stress the importance of differentiating between "true" gynecomastia and "lipomastia" (pseudogynecomastia), characterized by subcutaneous fat deposition. The importance of switching antiretroviral drugs is also discussed. In addition, newer medical therapies are highlighted.

Gynecomastia↗