Grantee Research Project Results
2001 Progress Report: Molecular Detection and Environmental Survey of Vegetative and Coccoid Helicobacter pylori
EPA Grant Number: R828037Title: Molecular Detection and Environmental Survey of Vegetative and Coccoid Helicobacter pylori
Investigators: Shahamat, Manouchehr , Bradley, Brian , Robb, Frank , Sowers, Kevin , Alavi, Mohammad
Current Investigators: Shahamat, Manouchehr , Gonzalez, Juan M , Bradley, Brian , Pavlick, Christine , Maeder, Dennis , Robb, Frank , Hind, John , Watts, Joy , Sowers, Kevin , Alavi, Mohammad
Institution: University of Maryland - Baltimore
Current Institution: University of Maryland Biotechnology Institute
EPA Project Officer: Hahn, Intaek
Project Period: May 15, 2000 through May 14, 2003
Project Period Covered by this Report: May 15, 2000 through May 14, 2001
Project Amount: $522,145
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objective of this research project is to develop and validate an accurate and sensitive molecular and/or immunological method for rapid detection of Helicobacter pylori in both the culturable and coccoid forms from environmental samples. This ability is crucial for preventing underestimation of the number of bacteria in samples, developing epidemiological evidence, and understanding the ecological and public health significance.
Progress Summary:
Four laboratory approaches have been followed in the development of H. pylori-specific molecular and immunological probes:
Intergenic Spacer Region (ISR). We have found a conserved DNA area downstream of the 16S rRNA gene in H. pylori strains. This region is unique to H. pylori strains and related Campylobacter species. Progress has been made in identifying those variable regions that can be used for high throughput screening. Sequencing of amplified fragments derived from several strains has identified a consistently unique DNA region in all strains. In addition, a consensus primer site has been identified. Evidence to date suggests that this variable region may provide a unique sequence that can be used to discriminate between individual strains of H. pylori.
Proteomics. We intend to use proteomics to distinguish between the vegetative and coccoid forms of H. pylori. Proteins from H. pylori strains were extracted and processed. Composite gels were created from original gel images and compared among strains and growth phases. The results indicate considerable differences among treatments. The unique spots represent 10-25 percent of the total spots in the range and sensitivity of the separation and staining. These results indicate that, of the proteins in the sample examined, there are few in common and many that are unique.
Indirect Fluorescent Antibody. Monoclonal antibody has been applied using IFA method and the preliminary results using IgG are promising.
DNA Probes. Specific DNA probes are being prepared and evaluated. Results indicated that our probes detected all 11 strains tested and did not amplify DNA from non-Helicobacter strains
Future Activities:
We will focus on obtaining a longer sequence for current strains in the study and sequences for more strains to determine if there is sufficient sequence variability in this region to allow discrimination of individual strains of H. pylori. We will use these probes to identify H. pylori from a mixture of genomic DNA from a variety of bacterial species. We will complete additional protein analysis to distinguish between vegetative and coccoid forms of H. pylori. We also will develop polymerase chain reaction (PCR) conditions for selective amplification of H. pylori strains by varying the probes design, buffer composition and cycling program to increase hybridization stringency. In addition, we will test additional strains in vegetative and coccoid forms with the monoclonal antibody. We will apply the antibody to different species spiked with H. pylori cells to determine the specificity and sensitivity of the IFA probe.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 7 publications | 1 publications in selected types | All 1 journal articles |
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Shahamat M, Alavi M, Watts JEM, Gonzalez JM, Sowers KR, Maeder DW, Robb FT. Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori. Journal of Clinical Microbiology 2004;42(8):3613-3619. |
R828037 (2001) R828037 (Final) |
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Supplemental Keywords:
Helicobacter pylori, coccoid forms, detection, evaluation, proteomics., RFA, Scientific Discipline, Water, Health Risk Assessment, Environmental Chemistry, Environmental Microbiology, Drinking Water, microbial contamination, coccoid heliobacter pylori, treatment, microbial risk assessment, fecal contamination, monitoring, environmental survey, exposure, immunological assay, molecular detection, bacteria, detection, public health, olgonucleotideProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.