Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation. The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated. During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly. Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling

Molecular identification of python species: Development and validation of a novel assay for forensic investigations

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (> 5500 samples, > 2.5 x 10(12) bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies

Distúrbios nutricionais em atletas femininas e suas inter-relações Nutrition disorders in female athletes and their interrelationships

Pesquisas indicam que é elevada a prevalência de distúrbios alimentares em atletas femininas jovens envolvidas em esportes que preconizam a magreza e o baixo peso corporal, tais como ginástica olímpica e corridas de longa distância. A fim de compreender a etiologia destes problemas, esta revisão apresenta os principais distúrbios nutricionais encontrados em atletas femininas, incluindo definições, critérios para diagnóstico, fatores de risco, presença de desordens inter-relacionadas - anemia, irregularidades menstruais e desmineralização óssea e suas conseqüências sobre a saúde e rendimento atlético. É fundamental ressaltar a importância da nutrição no desempenho físico. A presença de um baixo peso corporal e um consumo energético severamente restrito é o principal fator de risco para o desenvolvimento de tais distúrbios. Como as atletas e seus treinadores não apresentam adequados conhecimentos sobre nutrição, é necessária uma orientação nutricional individualizada, de forma a evitar o desenvolvimento destas alterações.<br>Researches have indicated that the prevalence of nutrition disorders is high among young female athletes engaged in sports that emphasize the importance of leanness and low body weight, such as olympic gymnastics and distance running. In order to understand the etiology of these problems, this review presents the main nutrition disorders in female athletes, including definitions, diagnostic criteria, risk factors, presence of interrelated disorders (anemia, menstrual irregularities and failure of bone mineralization) and their consequences on health and athletic performance. It is fundamental to point out the importance of nutrition on physical performance. The presence of a low body weight and a very restricted energy intake is the principal risk factor for the development of nutritional disturbances. The athletes and their coaches do not have an apropriate knowledge about nutrition, so it is necessary an individualized nutritional orientation to avoid the development of these disturbances