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Using a sensitive and rapid method combining broad-range PCR amplification of bacterial 16S rDNA fragments and pyrosequencing for detection, identification and typing, we have found contaminating bacterial DNA in our reagents used for PCR. Identified bacteria are the water-borne bacterial genera Pseudomonas, Stenotrophomonas, Xanthomonas, Ralstonia and Bacillus. Our results are in concordance with recent reports of contaminated industrial water systems. In light of this conclusion, we believe that there is a need for increased awareness of possible contamination in uncertified widely used molecular biology reagents, including ultra-pure water. Since sequencebased 16S rDNA techniques are used in a variety of settings for bacterial typing and the characterization of microbial communities, we feel that future certification of molecular biology reagents, as free of nucleic acids, would be advantageous.

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27 %). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P ¼ 0 . 815), nor between Dukes' classes A/B and C/D (P ¼ 0 . 262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens. INTRODUCTIONHelicobacter pylori is a microaerophilic Gram-negative spiral-shaped bacterium (Marshall & Warren, 1983) associated with chronic gastritis, peptic ulcer and gastric adenocarcinoma development (Parsonnet et al., 1991;Cover & Blaser, 1992;Logan, 1994). It has been shown that there is a significant geographical relationship between gastric cancer mortality rates and prevalence of H. pylori infection. Subtypes of H. pylori might differ in pathogenicity (Xiang et al., 1995), and a number of studies have shown that the prevalence of H. pylori expressing the cytotoxin-associated gene A (cagA) in gastric cancer patients is much higher than in age-and gender-matched controls (Parsonnet et al., 1997; Konturek et al., 2003;Semino-Mora et al., 2003). Recent reports imply that H. pylori may be an association factor involved in colorectal cancer (CRC) development in patients infected with H. pylori strains (Meucci et al., 1997;Shmuely et al., 2001). However, it is far from clear whether H. pylori is present in CRC tissues and whether or not H. pylori plays a similar role in colorectal carcinogenesis as has been proposed for gastric cancer development.16S rDNA sequence analysis has demonstrated considerable genomic diversity among H. pylori clinical isolates, and numerous sequence-specific PCR assays, combined with 16S rDNA sequencing, have been developed to identify Helicobacter species (Thoreson et al., 1995;Blom et al., 2002). We recently described a method for the rapid simultaneous molecular identification and subtyping of H. pylori by pyrosequencing analysis of the 16S rDNA variable V1 and V3 regions (Monstein et al., 2001).In this report we describe the molecular identification of Helicobacter DNA in CRC biopsies by means of a 16S rDNA PCR amplification assay combined with pyrosequencing analysis. METHODSTissue collection and DNA isolation. A total of 77 patients (45 w...

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Background: It has been shown that the non-opioid effects of Met-enkephalin, which is derived from proenkephalin-A, are mediated through a specific opioid growth factor (OGF) receptor which is assumed to be involved in the control of cell growth. The expression and tissue location of proenkephalin-A mRNA in the gastrointestinal tract remains largely unknown. Methods: In this study we have analyzed the expression of proenkephalin-A mRNA in the human esophagus, gastrointestinal tract and surrounding organs by means of reverse-transcriptase PCR (RT-PCR). Results: The present study demonstrates proenkephalin-A mRNA expression in the human esophagus, gastrointestinal tract, pancreas, and gallbladder. Conclusion: The present study demonstrates proenkephalin-A mRNA expression in regions of the human esophagus, gastrointestinal tract, pancreas, and gallbladder tissues which provides information for the future mapping of proenkephalin-A mRNA and protein expression/co-expression at the cellular level.