Micronutrients

Micronutrients are essential dietary components and play a fundamental role in the prevention of disease. 30 are essential and cannot be synthesized by the body on a daily basis, making dietary sources critical. Micronutrients have an array of biochemical functions which are fundamental in the homoeostatic regulation of body function. It is at any point in a metabolic pathway that the chemical reaction may be unable to continue along its nature cascading pathway because the essential micronutrient is lacking. The normal metabolic regulation of the body will have been disturbed and ill health may occur due to the absence of a specific micronutrients. Several population groups with specific nutritional requirements, complex social, environmental and economic circumstances may be at risk of inadequate micronutrient intakes due to poor consumption or excessive losses, and hence maybe unable to meet their recommended requirements of micronutrients. This group may benefit from micronutrient supplements. Infants/children are a specific population group who are at risk of micronutrient deficiency states making adequate intakes essential to ensure normal growth and development. The paediatric group will be discussed here, with reference to current recommendations for their micronutrient and supplementation requirements based on current evidence available to advisory groups.     

Direct sequence confirmation of qPCR products for gene doping assay validation in horses

The misuse of gene therapy by the introduction of transgenes via plasmid or viral vectors as a doping agent is an increasing concern in human and animal sports, not only in consideration to fair competition but also in potential detrimental effects to welfare. Doping events can be detected by polymerase chain reaction (PCR) amplification of a transgene-specific region of DNA. Quantitative real-time PCR (qPCR) is particularly suited to confirmatory investigations where precise limits of detection can be calculated. To fully validate a qPCR experiment, it is highly desirable to confirm the identity of the amplicon. Although post-PCR techniques such as melt curve and fragment size analysis can provide strong evidence that the amplicon is as expected, sequence identity confirmation may be beneficial as part of regulatory proceedings. We present here our investigation into two alternative processes for the direct assessment of qPCR products for five genes using next-generation sequencing: ligation of sequence-ready adapters to qPCR products and qPCR assays performed with primers tailed with Illumina flow cell binding sites. To fully test the robustness of the techniques at concentrations required for gene doping detection, we also calculated a putative limit of detection for the assays. Both ligated adapters and tailed primers were successful in producing sequence data for the qPCR products without further amplification. Ligated adapters are preferred, however, as they do not require re-optimisation of existing qPCR assays.