RESUMO
A pilot study was performed using two different DNA technology platforms conducted by two laboratories to analyze DNA extracted from 83-year-old, human male skeletal remains from 16 individuals, of which there are no other viable means to identify these war victims. The workflow of the more recent developed ForenSeq Kintelligence Kit and next generation sequencing was compared to that of the standard capillary electrophoresis - short tandem repeat (STR) method (Power Plex ESX17 and Y23 Systems). The findings indicate that greater amount of useful genetic data can be gained with the Kintelligence system across the range of samples under study and particularly for samples in which partial or no STR profiles are obtained. SNP data are more likely to be obtained from degraded samples, like the ones analyzed in this study. Moreover, high volume SNP data are suitable for long distance kinship associations and genetic genealogy databases to develop more investigative leads for future kinship and missing persons cases, a process not feasible by STR typing.
RESUMO
A label-free biosensor based on white light reflectance spectroscopy for the determination of PSA as semen indicator in forensic samples is presented. The sensor is based on a two-step immunoassay which employs the same polyclonal anti-PSA antibody as capture and detection antibody followed by reaction with streptavidin as a signal enhancement step. The whole assay time was set to 10min; 5min reaction of immobilized antibody with the PSA calibrators or the samples, 3min reaction with the biotinylated anti-PSA antibody and 2min reaction with streptavidin. Following this protocol, a detection limit of 0.5ng/mL was achieved and the assay's linear response range extended up to 500ng/mL. Thus, taking into account the quantification limit of 1.0ng/mL and the average PSA concentration in semen (0.2-5.5mg/mL), semen quantities of a few nanoliters could be detected. The accuracy of the sensor developed was demonstrated through recovery (% recovery ranged from 89.6 to 106) and semen dilution experiments. A linear correlation was found for semen dilutions ranging from 5000 to 360,000. The lack of interference by other bodily fluids was confirmed by analysing stains of blood, urine and saliva prior to and after the addition of semen. Finally, the sensor was evaluated by analysing 51 forensic casework samples which were also analysed with a semi-quantitative membrane strip test (Seratec® PSA), through microscopic detection of spermatozoa, and male DNA identification through detection of Y chromosome. The results obtained with the sensor were in excellent agreement with those provided by an immunoradiometric assay kit (PSA-RIACT) and in complete agreement with the findings using the membrane strip assay, spermatozoa and Y chromosome detection. The excellent analytical performance and small size of the instrument make the sensor developed an attractive tool for use in forensic evidence screening for semen detection.
