DNA methylation ratio variability may impede clinical application of cancer diagnostic markers.

Hypermethylation at promoter regions of tumour suppressor genes is diagnostic for many cancers. Many genomic regions that may be the targets for clinical diagnostic assays have been identified through use of measuring systems reliant on bisulphite conversion, but few of these promising markers are in clinical use. The comparability of a widely used DNA methylation measuring system involving bisulphite conversion was evaluated by supplying three experienced centres with methylated DNA reference material mixtures that were independently prepared and characterised by mass spectrometry and high-pressure liquid chromatography. A replication scheme was designed to evaluate reproducibility of key analytical steps within and between laboratories by regression analysis. In general, methylation was underestimated and methylation ratio values were highly variable. The difference in methylation ratio between CpG sites was the key contributor to variable results. The CpG site effect followed a similar pattern at all centres and at all methylation levels examined indicating that sequence context had a major effect on methylation ratio measurement using the bisulphite conversion process. The magnitude of underestimation combined with the variability of measurements between CpG sites compromises the concept of measuring genomic regional methylation by averaging the methylation ratios of many CpG sites. There were no significant differences in replicate bisulphite conversions or sample work-up and instrument analysis at each centre thus making this technique suitable for comparative intralaboratory investigations. However, it may not be suitable for a routine diagnostic assay without extensive standardisation efforts.

Improved detection of transgene and nonviral vectors in blood.

Vector biodistribution and clearance studies are essential in the development of gene transfer medicine. To provide reliable and accurate data, protocols for vector analysis must be optimized and validated. We addressed several parameters affecting the detection of gene therapy vectors in blood. Using an in vitro system based on plasmid DNA incorporating, as a transgene, complementary DNA for human erythropoietin gene, we developed and validated a suite of real-time PCR assays for the transgene splicing sites. The most sensitive assays detected the transgene present at 0.011% of the copy number of the endogenous erythropoietin gene in human genomic DNA at 100% specificity. Plasmid linearization incorporated with PCR resulted in an increase in assay sensitivity up to 4.5-fold without compromising analysis workflow. This allowed detection of five copies of transgene in a background of 0.4 µg of genomic DNA (or 0.0035% detectable transgene copies relevant to copies of the endogenous gene). Finally, desktop assessment of 18 DNA extraction protocols was undertaken and 5 kits were evaluated experimentally for extraction of nonviral vectors from blood. Three kits reliably detected 80 copies of the transgene in a milliliter of blood. Adoption of the described protocols will enable more reliable vector analysis in gene therapy and will assist in accurate interlaboratory comparison. The methodology will also facilitate detection of gene doping in sport, a potential new form of misuse of gene transfer technology.

Detection of Helicobacter species in the gastrointestinal tract of ringtail possum and koala: possible influence of diet, on the gut microbiota.

The presence of Helicobacter spp. was examined in the liver and in different regions of the gastrointestinal tract (GIT) including the stomach, 3 cm above ileum, ileum, caecum, colon and rectum of 10 ringtail possums (RTPs) and 3 koalas using a combination of microscopy, culture and PCR. Helicobacter was detected in the distal end of the GIT of 7 of 10 RTPs by direct PCR and in all (10/10) RTPs by nested PCR. Five 'S' shaped isolates with bipolar sheathed flagella were isolated from the lower bowel of 3 of the 10 RTPs. 16S rRNA sequence analysis of these 5 isolates confirmed them as potentially novel Helicobacter species. No Helicobacter species were cultured from the koalas, however Helicobacter DNA was detected, in the majority of liver and/or stomach samples of the three koalas and in the colonic region of one koala, using nested PCR. The 16S rRNA gene was sequenced directly from DNA extracted from the homogenised livers and mucus scrapings of the stomach from koala 1 and were confirmed to be Helicobacter species. Based on histopathological examination of sections from the liver and intestine no evidence of infection could be related to the presence of helicobacters in either the RTP or koala. Based on our results, it is possible that diet may influence the detection of Helicobacter species; however this required further investigation.

Detection, isolation, and characterization of helicobacter species from the gastrointestinal tract of the brushtail possum.

The presence of Helicobacter species in Australian marsupials was examined systematically using microscopy, culture, and PCR in different regions of the gastrointestinal tract (GIT) and in the liver of brushtail possums (BTPs) (Trichosurus vulpecula), a common Australian marsupial that feeds on eucalyptus leaves. The spatial distribution of Helicobacter species in the GIT sections also was examined microscopically in silver-stained sections and by fluorescent in situ hybridization (FISH) using a Helicobacter genus-specific probe. Helicobacter species were found colonizing the lower bowel of all BTPs studied. Good agreement was observed between the detection of Helicobacter species using culture and PCR, which was supported by the microscopic examination of silver-stained sections and FISH. The lower bowel of BTPs were colonized by one to three morphologically different (a comma-shaped species with no apparent flagella, a fusiform-shaped species entwined with periplasmic fibers and a bipolar sheathed flagella, and an S-shaped species with bipolar sheathed flagella) and potentially novel Helicobacter species, as well as in one case with a potentially novel Campylobacter species, which was a tightly coiled rod with bipolar unsheathed flagella. The isolation and characterization of these Helicobacter species in BTPs provides important information regarding the specific natural niche of these bacteria and their corelationship within their host, and it increases our understanding of the ecology of Helicobacter species.