Concurrent genotyping of Helicobacter pylori virulence genes and human cytokine SNP sites using whole genome amplified DNA derived from minute amounts of gastric biopsy specimen DNA

<p>Abstract</p> <p>Background</p> <p>Bacterial and cellular genotyping is becoming increasingly important in the diagnosis of infectious diseases. However, difficulties in obtaining sufficient amount of bacterial and cellular DNA extracted from the same human biopsy specimens is often a limiting factor. In this study, total DNA (host and bacterial DNA) was isolated from minute amounts of gastric biopsy specimens and amplified by means of whole genome amplification using the multiple displacement amplification (MDA) technique. Subsequently, MDA-DNA was used for concurrent <it>Helicobacter pylori </it>and human host cellular DNA genotyping analysis using PCR-based methods.</p> <p>Results</p> <p>Total DNA was isolated from gastric biopsy specimens of 12 subjects with gastritis and 16 control subjects having a normal mucosa. The DNA was amplified using a multiple displacement amplification (MDA) kit. Next, concurrent genotyping was performed using <it>H. pylori</it>-specific virulence gene PCR amplification assays, pyrosequencing of bacterial 16S rDNA and PCR characterisation of various host genes. This includes Interleukin 1-beta (<it>IL1B</it>) and Interferon-gamma receptor (<it>IFNGR1</it>) SNP analysis, and Interleukin-1 receptor antagonist (<it>IL1RN</it>) variable tandem repeats (VNTR) in intron 2. Finally, regions of the <it>vacA</it>-gene were PCR amplified using M13-sequence tagged primers which allowed for direct DNA sequencing, omitting cloning of PCR amplicons. <it>H. pylori </it>specific multiplex PCR assays revealed the presence of <it>H. pylori cagA </it>and <it>vacA </it>genotypic variations in 11 of 12 gastritis biopsy specimens. Using pyrosequencing, 16S rDNA variable V3 region signatures of <it>H. pylori </it>were found in 11 of 12 individuals with gastritis, but in none of the control subjects. Similarly, <it>IL1B </it>and <it>IFNGR1</it>-SNP and <it>IL1RN</it>-VNTR patterns could be established in all individuals. Furthermore, sequencing of M13-sequence tagged <it>vacA</it>-PCR amplicons revealed the presence of highly diverse <it>H. pylori vacA</it>-s/i/m regions.</p> <p>Conclusion</p> <p>The PCR-based molecular typing methods applied, using MDA-amplified DNA derived from small amounts of gastric biopsy specimens, enabled a rapid and concurrent molecular analysis of bacterial and host genes in the same biopsy specimen. The principles and technologies used in this study could also be applied to any situation in which human host and microbial genes of interest in microbial-host interactions would need to be sequenced.</p

Cloning and expression of two different genes from Streptococcus dysgalactiae encoding fibronectin receptors.

Binding of bacteria to fibronectin has been implicated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcus dysgalactiae to fibronectin. The cells bind to a site in the NH2-terminal domain of the protein via trypsin-sensitive cell surface components. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding proteins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with 125I-labeled fibronectin, a 140-kDa fibronectin-binding protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibronectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction mapping and cross-hybridization experiments. The two encoded proteins were also immunologically distinct although both bound to the same region of the fibronectin molecule, and both effectively inhibited the binding of 125I-fibronectin to bacterial cells. Immunological analyses showed that only one of the two proteins tentatively identified as fibronectin receptors was expressed in detectable quantities in the Streptococcus dysgalactiae strain under the culture conditions employed

Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

<p>Abstract</p> <p>Background</p> <p>Plasmid encoded ^<it>bla</it>CTX-M enzymes represent an important sub-group of class A β-lactamases causing the ESBL phenotype which is increasingly found in <it>Enterobacteriaceae </it>including <it>Klebsiella </it>spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.</p> <p>Methods</p> <p>Multiple displacement amplified DNA derived from 20 <it>K. pneumoniae </it>and 34 <it>K. oxytoca </it>clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.</p> <p>Results</p> <p>Nine out of 20 <it>K. pneumoniae </it>clinical isolates had a ^<it>bla</it>CTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 <it>K. oxytoca </it>clinical isolates. Molecular identification and differentiation between ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. <it>In silico </it>analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located ^<it>bla</it>OXY and K1-genes in <it>Klebsiella </it>spp. and K1-like genes in other <it>Enterobacteriaceae</it>.</p> <p>Conclusion</p> <p>The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of ^<it>bla</it>CTX-M, and ^<it>bla</it>OXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.</p

Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs

<p>Abstract</p> <p>Background</p> <p>The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of <it>Helicobacter pylori </it>has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of <it>cagA </it>EPIYA motifs.</p> <p>Findings</p> <p>MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the <it>cagA </it>EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the <it>cagA </it>EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) <it>cagA </it>EPIYA motif, respectively. In two cases, double <it>cagA </it>EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two <it>H. pylori </it>strains in the same biopsy.</p> <p>Conclusion</p> <p>Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of <it>cagA </it>EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed <it>H. pylori </it>strains present in the same biopsy specimen.</p

Open Access

Expression of multiple forms of 3&apos;-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas

Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinoma

BACKGROUND: Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. FINDINGS: PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. CONCLUSION: Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs

Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas

Abstract Background Two main types of receptors for gastrin and cholecystokinin (CCK) have been cloned and identified. CCK1 (CCK-A) receptors are expressed in the pancreas, the gallbladder, and parts of the brain, while CCK2 (CCK-B/gastrin) receptors (CCK2R) are expressed in gastric glands and in most of the brain. A splice variant of the CCK2R designated CCKRi4sv (CCK-C), which is constitutively expressed in human pancreatic cancer cells, has also been described. The purpose of the present investigation was to study CCK2R, CCK2i4svR, and gastrin mRNA expression in human pancreatic adenocarcinoma on the assumption that co-expression of CCK2R and gastrin or constitutive CCK2i4svR mRNA expression plays a pivotal role in the progression of pancreatic cancer. Findings PCR amplification using CCK2R specific primer-pairs, followed by ethidium-bromide stained agarose gel electrophoresis revealed the expression of wild-type CCK2R mRNA in 12 of 17 biopsy specimens. A CCK2R intron 4 specific nested PCR assay revealed that CCK2i4svR mRNA was expressed in only one of the biopsy specimen. The authenticity of PCR amplicons was confirmed by cloning of selected amplicons and DNA sequence analysis. Moreover, we found that hitherto undescribed multiple forms of 3'-end variant CCK2R mRNAs with various deletions in the retained intron 4 and exon 5, tentatively generating truncated proteins, were expressed in the pancreatic adenocarcinomas. Conclusion Cloning and DNA sequencing of selected amplicons revealed that CCK2R and multiple CCK2i4svR-like mRNAs are expressed in human pancreatic adenocarcinoma. The originally described CCK2i4svR mRNA was only expressed in one of 17 tumours and appears to be rarely expressed in pancreatic adenocarcinoma. We report that CCK2R- and gastrin mRNA co-expression may play a role in a portion, but not in all of these tumours, and that aberrant splicing takes places in these tissues generating multiple forms of 3'-end variant CCK2R mRNAs.</p