Design and Optimization of a Mycoplasma Detection Assay

Mycoplasma are among the smallest free living microorganisms. These bacteria grow slowly, lack a rigid cell wall and are not eliminated by filter sterilization methods used in tissue culture. Mycoplasma infection affects biochemical and genetic aspects of cultured cells, resulting in experimental inconsistency. Therefore, it is necessary to establish routine testing for mycoplasma contamination in tissue culture laboratories. Our goal is to develop a reliable and cost-effective test for mycoplasma in cell culture based on established methods found in literature. We first cloned and sequenced a PCR product from a commercial mycoplasma detection kit. Sequencing revealed the 16s rRNA as the target for mycoplasma detection; we confirmed this target by conducting a literature search. PCR primers were designed using 16s rRNA gene as a target. We set-up reactions and optimized conditions for the real-time PCR assay to detect the target and confirmed amplicon size with agarose gel electrophoresis. We identified that 56oC was the best temperature for the PCR and found that agarose gel electrophoresis was a better detection method because it identified the size to confirm the proper product. The primers we ordered to develop this assay produce the proper band; however, results of several assays have been inconsistent as sometimes a known positive sample fails to amplify. As well, in several PCR reactions the negative showed a signal. The overall reaction needs improvements to have greater reliability and to eliminate all sources of contamination. Research is continuing results are not final

HIV-1 Vpr Causes Synaptodendritic Damage in Neurons

HIV weakens the immune system by infecting and destroying T-cells, leaving the body vulnerable to infection and the development of AIDS. Conventional treatments for HIV, such as combined anti-rectroviral therapy (cART), fail to prevent the development of HIV-associated neurocognitive disorder (HAND). Neurological dysfunction has been directly related to the invasion of HIV in the central nervous system (CNS). HIV produces neurotoxic proteins, such as the Viral Protein R (Vpr), which contribute to HAND. Astrocytes are the most abundant cells in the brain and an important HIV target. We hypothesize that astrocytes expressing Vpr will cause neuronal damage in our co-culture system. Primary astrocytes were transfected with Vpr plasmid or control (pEGFP or mock) using electroporation. Astrocytes were then co-cultured with cortical neurons. At 48 and 72 hours we collected the primary astrocytes to confirm the Vpr expression via western blot analysis. We then measured structural damage in the neurons using immunofluorescence for cytoskeletal (MAP2, f-actin) and synaptic (synaptophysin) damage. Preliminary results showed strong staining of filamentous actin and MAP2 with weak detection of synaptophysin. The positive control for neurotoxicity (2.8µM acrylamide) showed substantial damage to the cellular structure. Results for Vpr expression are pending. After confirming that the immunofluorescence assays are working with our controls, we expect to detect any synaptodendritic damage in the neurons caused by Vpr in our upcoming experiments