Development of HyBeacon® probes for specific mRNA detection using body fluids as a model system.

HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods. The work presented here details the development of a single-step one-tube RT-PCR assay to detect the presence of body fluids of forensic interest (saliva, blood, seminal fluid, vaginal fluid and menstrual blood) using HyBeacon® probes and melt curve analysis. Each assay shows a high degree of specificity to the target body fluid mRNA suggesting there is no requirement to remove genomic DNA prior to analysis. Of the five assays developed, four were able to detect between 10 and 100 copies of target cDNA, the fifth 1000 copies of target. The results presented here demonstrate that such an approach can be optimised for non-expert users and further areas of work are discussed.

Developmental validation of the ParaDNA(®) Intelligence System-A novel approach to DNA profiling.

DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum.

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37°C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS-PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3-4 h at 37°C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20°C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37°C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.