39 research outputs found

    Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

    Get PDF
    BACKGROUND: The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. METHODS: Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng) diluted in a background of C-33A DNA (100 ng-2 μg). Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. RESULTS: HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16) in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Multiple infections were also detectable using a touchdown approach. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. CONCLUSION: Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity

    Chromosomal microarray testing in adults with intellectual disability presenting with comorbid psychiatric disorders.

    Get PDF
    Chromosomal copy-number variations (CNVs) are a class of genetic variants highly implicated in the aetiology of neurodevelopmental disorders, including intellectual disabilities (ID), schizophrenia and autism spectrum disorders (ASD). Yet the majority of adults with idiopathic ID presenting to psychiatric services have not been tested for CNVs. We undertook genome-wide chromosomal microarray analysis (CMA) of 202 adults with idiopathic ID recruited from community and in-patient ID psychiatry services across England. CNV pathogenicity was assessed using standard clinical diagnostic methods and participants underwent comprehensive medical and psychiatric phenotyping. We found an 11% yield of likely pathogenic CNVs (22/202). CNVs at recurrent loci, including the 15q11-q13 and 16p11.2-p13.11 regions were most frequently observed. We observed an increased frequency of 16p11.2 duplications compared with those reported in single-disorder cohorts. CNVs were also identified in genes known to effect neurodevelopment, namely NRXN1 and GRIN2B. Furthermore deletions at 2q13, 12q21.2-21.31 and 19q13.32, and duplications at 4p16.3, 13q32.3-33.3 and Xq24-25 were observed. Routine CMA in ID psychiatry could uncover ~11% new genetic diagnoses with potential implications for patient management. We advocate greater consideration of CMA in the assessment of adults with idiopathic ID presenting to psychiatry services

    Streshanteringsopleiding van 'n risikogroep

    Get PDF
    M.Cur. (Curriculum Studies)The aim of this study is to determine whether training in stress management through primary mental health education has a positive effect on the learning of effective stress management techniques. This study was carried out on a group of police officers. In an experimental investigation the following method of stress management was used, namely a primary mental health education programme. An at-risk population in the society was identified namely the South African Police Force, and sixty four people from the target population of one hundred and twenty eight candicate-officers 'were selected for this study. A survey of the target population was done and these sampling units met the following criteria: they were all white warrant officers in training in the South.African Police Force from the same level of training, who had all obtained a national diploma in police administration. These members were representatives from throughout South-Africa making it a national study. Their ages ranged from twenty to fifty years of age...

    Conserving koalas: A review of the contrasting regional trends, outlooks and policy challenges

    No full text
    The koala (Phascolarctos cinereus), one of the world's most iconic faunal species, was recently listed under Australian government legislation as vulnerable in the northern states of Queensland and New South Wales and in the Australian Capital Territory

    Conserving koalas a review of the contrasting regional trends, outlooks and policy challenges /

    No full text
    The koala (Phascolarctos cinereus), one of the world's most iconic faunal species, was recently listed under Australian government legislation as vulnerable in the northern states of Queensland and New South Wales and in the Australian Capital Territory, but not in the southern states of Victoria and South Australia. This review synthesises empirical evidence of regional koala population trends, their conservation outlook, and associated policy challenges. Population declines are common in the northern half of the koala's range, where habitat loss, hotter droughts, disease, dog attacks and vehicle collisions are the major threats. In contrast, some southern populations are locally overabundant and are now subject to managed declines. The koala presents the problem of managing a wide-ranging species that now primarily occurs in human-modified landscapes, some of which are rapidly urbanising or subject to large-scale agricultural and mining developments. Climate change is a major threat to both northern and southern populations. The implementation of policy to conserve remaining koala habitat and restore degraded habitat is critical to the success of koala conservation strategies, but habitat conservation alone will not resolve the issues of koala conservation. There needs to be concerted effort to reduce the incidence of dog attack and road-related mortality, disease prevalence and severity, and take into account new threats of climate change and mining. Many of the complex conservation and policy challenges identified here have broader significance for other species whose population trends, and the nature of the threatening processes, vary from region to region, and through time
    corecore