Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis.

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

Subtyping of Listeria monocytogenes isolates recovered from retail ready-to-eat foods, processing plants and listeriosis patients in Sweden 2010.

Identification and prioritisation of food safety interventions requires an understanding of the relationship between food, pathogens and cases. Such understanding can be gained through different approaches, e.g. microbial subtyping to attribute cases of foodborne disease to food vehicles or other sources of illness. In this study, Listeria monocytogenes isolates (n=166) from (i) three categories of ready-to-eat (RTE) foods, (ii) food processing plant environments, and (iii) human listeriosis cases, all sampled during 2010 in Sweden, were subtyped. In addition, 121 isolates from human listeriosis cases, collected 2005-2009, were subtyped. Subtyping consisted of both serotyping (conventional method and PCR) and genotyping using pulsed-field gel electrophoresis (PFGE). Serotype 1/2a dominated in all three groups of isolates (range 73-96%). Eighteen percent of the human isolates (2010) belonged to serotype 4b, but only 1.4% of the food isolates. The food isolates differentiated into 19 pulsotypes (ID=0.843), the human isolates collected 2010 into 31 pulsotypes (ID=0.950) and the processing plant isolates into 22 pulsotypes (ID=0.991). Six of the pulsotypes were shared between the food and human isolates. These pulsotypes comprised 42% of the human isolates and 59% of the food isolates. For some processing plants, there was suggested persistence of one or more specific L. monocytogenes strains, as indicated by repetitive isolation of the same pulsotype from food. This study indicated the presence of L. monocytogenes in the processing plant environment as a likely source of contamination of gravad and cold-smoked fish, and this food category as an important source of human exposure to the pathogen.