Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Germination and Photosynthetic Responses to Salinity and Alkalinity in <i>Avicenna marina</i> Propagules

Avicenna marina (Forssk.) Vierh is a halophytic mangrove. The reproductive unit is green and has photosynthetic propagules. Mangroves are naturally exposed to fluctuations in some abiotic factors at the soil surface, including salinity and alkalinity. The objective of this study was to determine the effects of two salts including NaCl and NaHCO₃ on germination processes and discuss the relationships between cotyledon photosynthesis and embryo axis growth in A. marina propagules. These propagules came from Al Birk, located on the shoreline of the Saudi Red Sea. The results showed that the studied salts did not affect neither the final germination percentage nor the embryo axis growth. However, rooting and root growth were delayed by both salts at 300 mM and were strongly inhibited by 600 mM NaHCO₃. Both NaCl and NaHCO₃ reduced the photosynthetic activity. These two salts did not affect the other photosynthetic parameters, including stomatal conductance, net transpiration, and intercellular CO₂. Thereafter, the reduction in net photosynthesis was not related to any limitation of stomatal conductance. The early germination phase was independent of cotyledon photosynthesis, whereas rooting and root growth may be limited by reduced photosynthesis under NaCl and NaHCO₃.     

Histochemical Localization of Essential Oils and Bioactive Substances in the Seed Coat of the Halophyte <Emphasis Type="Italic">Crithmum maritimum</Emphasis> L. (Apiaceae)

Despite seeds and fruits of several halophytes being rich in essential oils and other bioactive substances, the histochemical characterization of these compounds has not received much attention. The aims of the present investigation were to localize the essential oils and the bioactive substances in the seed coat of the oilseed halophyte Crithmum maritimum L. Fruits were collected from the rocky coasts of Tabarka (NW of Tunisia, 36°57′12″ N, 08°45′18″ E). C. maritimum L. seed is shown to be surrounded by two envelopes: The first structure is a secretory envelope, consisting in about 20 canals. The second layer represents the endocarp. As revealed by fluorescence and light microscopy, the essential oils, O-dihydroxyphenols and flavonoids, known as bioactive compounds, were accumulated in the canals. The endocarp layer accumulated polysaccharides, O-dihydroxyphenols, and flavonoids. As a whole, these findings highlight the histochemical features and confirm the valuable quality of C. maritimum L. seeds.