Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR.

Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze-thaw method, a freeze-boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.

A plasmid immunization construct encoding urease B of Helicobacter pylori induces an antigen-specific antibody response and upregulates the expression of beta-defensins and IL-10 in the stomachs of immunized mice.

The objectives of this study were to investigate the efficacy of a prototype DNA immunization construct encoding the urease B subunit enzyme of Helicobacter pylori (H. pylori) for inducing adaptive and innate immune responses in mice immunized via intramuscular or subcutaneous routes and to further explore the adjuvant effects of the CpG motifs in the vector. Antibody, cytokine, and beta-defensin profiles were assessed in the stomachs of immunized animals: experiments were terminated 3 months after immunization because there was a significant increase in the anti-H. pylori urease B antibody response at Week 6 in mice immunized with the urease B construct. A long lasting expression of IL-10 mRNA was noted. Furthermore, a marked and sustained increase in the mRNA expression of beta-defensins was also observed, particularly beta1. This study demonstrates that an H. pylori urease B DNA construct can induce innate as well as adaptive immune responses in the stomachs of immunized mice. Upregulation of beta-defensin gene expression followed immunization and we believe that this is the first report of a DNA vaccine inducing innate anti-microbial responses. Such complex molecular interactions that modulate both innate and adaptive immune responses may be of critical importance in the control of mucosal pathogens, such as H. pylori.