tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.
Detection and identification of body fluids are crucial aspects of forensic investigations, aiding in crime scene reconstructions and providing important leads. Although many methods have been developed for these purposes, no method is currently in use in the forensic field that allows rapid, non-contact detection and identification of vaginal fluids directly at the crime scene. The development of such technique is mainly challenged by the complex chemistry of the constituents, which can differ between donors and exhibits changes based on woman’s menstrual cycle. The use of fluorescence spectroscopy has shown promise in this area for other biological fluids. Therefore, the aim of this study was to identify specific fluorescent signatures of vaginal fluid with fluorescence spectroscopy to allow on-site identification. Additionally, the fluorescent properties were monitored over time to gain insight in the temporal changes of the fluorescent spectra of vaginal fluid. The samples were excited at wavelengths ranging from 200 to 600 nm and the induced fluorescence emission was measured from 220 to 700 nm. Excitation and emission maps (EEMs) were constructed for eight donors at seven time points after donation. Four distinctive fluorescence peaks could be identified in the EEMs, indicating the presence of proteins, fluorescent oxidation products (FOX), and an unidentified component as the dominant contributors to the fluorescence. To further asses the fluorescence characteristics of vaginal fluid, the fluorescent signatures of protein and FOX were used to monitor protein and lipid oxidation reactions over time. The results of this study provide insights into the intrinsic fluorescent properties of vaginal fluid over time which could be used for the development of a detection and identification method for vaginal fluids. Furthermore, the observed changes in fluorescence signatures over time could be utilized to establish an accurate ageing model.
This report presents the highlights of the 7th European Meeting on Molecular Diagnostics held in Scheveningen, The Hague, The Netherlands, 12-14 October 2011. The areas covered included molecular diagnostics applications in medical microbiology, virology, pathology, hemato-oncology,clinical genetics and forensics. Novel real-time amplification approaches, novel diagnostic applications and new technologies, such as next-generation sequencing, PCR lectrospray-ionization TOF mass spectrometry and techniques based on the detection of proteins or other molecules, were discussed. Furthermore, diagnostic companies presented their future visions for molecular diagnostics in human healthcare.
