Browsing by Subject "DNA extraction"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Open Access
    9β Polymorphism of the Glucocorticoid Receptor Gene Appears to Have Limited Impact in Patients with Addison’s Disease
    (Public Library of Science, 2014) Ross, Ian Louis; Dandara, Collet; Swart, Marelize; Lacerda, Miguel; Schatz, Desmond; Blom, Dirk Jacobus
    BACKGROUND: Addison’s disease (AD) has been associated with an increased risk of cardiovascular disease. Glucocorticoid receptor polymorphisms that alter glucocorticoid sensitivity may influence metabolic and cardiovascular risk factors in patients with AD. The 9β polymorphism of the glucocorticoid receptor gene is associated with relative glucocorticoid resistance and has been reported to increase the risk of myocardial infarction in the elderly. We explored the impact of this polymorphism in patients with AD. Materials and METHODS: 147 patients with AD and 147 age, gender and ethnicity matched healthy controls were recruited. Blood was taken in a non-fasted state for plasma lipid determination, measurement of cardiovascular risk factors and DNA extraction. RESULTS: Genotype data for the 9β polymorphism was available for 139 patients and 146 controls. AD patients had a more atherogenic lipid profile characterized by an increase in the prevalence of small dense LDL (p = 0.003), increased triglycerides (p = 0.002), reduced HDLC (p<0.001) an elevated highly sensitive C-reactive protein (p = 0.01), compared with controls. The 9β polymorphism (at least one G allele) was found in 28% of patients and controls respectively. After adjusting for age, gender, ethnicity, BMI and hydrocortisone dose per metre square of body surface area in patients, there were no significant metabolic associations with this polymorphism and hydrocortisone doses were not higher in patients with the polymorphism. CONCLUSIONS: This study did not identify any associations between the 9β polymorphism and cardiovascular risk factors or hydrocortisone dose and determination of this polymorphism is therefore unlikely to be of clinical benefit in the management of patients with AD.
  • Thumbnail Image
    Item
    Open Access
    Detection of Streptococcus pneumoniae from different types of nasopharyngeal swabs in children
    (Public Library of Science, 2013) Dube, Felix S; Kaba, Mamadou; Whittaker, Elizabeth; Zar, Heather J; Nicol, Mark P
    BACKGROUND: A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs. METHODS: The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA -targeted real-time polymerase chain reaction (qPCR). RESULTS: Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5-16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10 4 CFU/mL [IQR, 2.0×10 2 - 4.0×10 5 CFU/mL]) than Dacron swabs (3.7×10 4 CFU/mL [IQR, 4.0×10 2 -3.2×10 5 CFU/mL], p  = 0.17). Using lytA -targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10 5 genome copies/mL [IQR, 1.3×10 2 −1.8×10 6 ] vs. 9.3×10 4 genome copies/mL [IQR, 7.0×10 1 −1.1×10 6 ]; p  = 0.005). CONCLUSION: Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.
  • Thumbnail Image
    Item
    Open Access
    gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples
    (Public Library of Science, 2015) Vezzulli, Luigi; Stauder, Monica; Grande, Chiara; Pezzati, Elisabetta; Verheye, Hans M.; Owens, Nicholas J. P.; Pruzzo, Carla
    The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V . cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V . cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V . cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V . cholerae and failed to amplify strains of the closely-related species Vibrio mimicus . The sensitivity of the assay applied to environmental and stool samples spiked with V . cholerae ATCC 39315 was comparable to that of pure cultures and was of 10 2 genomic units/l for drinking and seawater samples, 10 1 genomic units/g for sediment and 10 2 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V . cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V . cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.