Progress 06/01/05 to 09/30/08
Outputs
OUTPUTS: Studies performed under the proposed aims of this project included the development and characterization of aptamers against salmonellae. The output of these studies will significantly enhance our ability to concentrate and detect salmonellase from complex food matrices. The findings of theses studies have been shared with peers at scientific fora.
PARTICIPANTS: Dr. Lee-Ann Jaykus, North Carolina State University
PROJECT MODIFICATIONS: None
Impacts
High affinity single stranded DNA aptamer ligands were generated against the purified outer membrane protein (OMP) of Salmonella Typhimurium using the combinatorial SELEX procedure. After seven rounds of SELEX, the pool was evaluated for specificity using Electrophoretic Mobility Shift Analysis. Upon positive determination of specificity of the pool, individual aptamers were cloned and evaluated in whole cell capture analysis by coupling biotinylated aptamers to streptavidin coated magnetic beads. Two aptamer candidates, aptamer 33 and aptamer 45 showed high sensitivity and were further evaluated using a wide array of techniques. Both the aptamers were 96 bases in length and consisted of a 40 mer randomized region with PCR primers flanking at the 5' and 3' end. These aptamers showed specific binding to three proteins out of a suite of proteins with varying molecular weights in the OMP when evaluated by South Western Blot(SW blot) analysis. Extensive analysis of the
protein binding by the aptamers was performed using the DNase footprinting assay. The randomized region of both the aptamers were observed to bind the OMP specifically and with high affinity. No binding was evident to a non specific protein, Bovine Serum Albumin. Spike and recovery of S. Typhimurium whole cells in bovine fecal samples displayed a sensitivity of approximately 10-40 CFU/gm when analyzed using a 119 bp DNA fragment specific to S.Typhimurium. from DNA extracted of the capture using Real Time PCR. The aptamers also showed a pan Salmonella specificity and a sensitivity of 100 CFU/ml in pure cultures for seven other strains, S. Dublin, S. Enteritidis, S. Fresno, S. Hadar, S. Javiana, S. Moscow, S. Newport. Conclusions: High affinity DNA aptamers capture and concentrate low numbers of Salmonella from complex matrices have been identified. These aptamers have been characterized using multiple techniques for specificity and sensitivity which is between 10-40 organisms/g bovine
species. Sensitive detection has been possible in the absence of culture based pre-enrichment steps. Future Outlook: These aptamers can be used in the sensitive detection of S. Typhimurium specifically from complex matrices. More matrices can be evaluated to understand the dynamics of matrix interference for aptamer based capture and concentration of Salmonella. These aptamers can be used potentially as pan Salmonella ligands and then coupled to down stream techniques such as serovar specific PCR for specific detection.
Publications
- Joshi, R Lee-Ann Jaykus, H. Janagama, J. Schefers, and S. Sreevatsan. 2007, DNA Aptamer Based Capture and Detection of Salmonella enterica serovar Typhimurium. Poster presented at the Annual Points of Pride Research Days, University of Minnesota, St Paul, MN. 20-21 March 2007. Summer Research Days At College of Veterinary Medicine.
- Joshi R, Sreevatsan S, 2007, DNA aptamers as tools for pre-analytical sample processing of complex matrices for faster detection of Salmonella enterica serovar Typhimurium, abs 164. Presented at the 88th conference of research workers for animal disease, Chicago, IL.
- Invention Discosure: S. Sreevatsan, 2007, Aptamers against salmonellae. University of Minnesota. Patent pending.
|
Progress 01/01/06 to 12/31/06
Outputs
OBJECTIVES: ssDNA aptamers identified against Salmonella Typhimurium outer membrane can be used as high affinity ligands to capture whole cell Salmonellae. APPROACH: DNA aptamers were selected to bind outer membrane preparations (OMPs) of Salmonella enterica serovar Typhimurium using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) using a 40-mer randomized library. A counter-SELEX approach was employed to eliminate aptamers cross-reactive to lipopolysaccharides (LPS) and OMPs of a closely related enterobacterium, E. coli. Gel shift analysis was used to validate the binding of selected aptamers to the OMPs and Real Time PCR assay for a 119 bp invA gene was use to detect the capture of Salmonella by the aptamers. SIGNIFICANT FINDINGS/ IMPACTS TO DATE: The SELEX process yielded a total of 66 aptamer candidates. Specificity of the aptamers was evaluated by gel-shift analysis against Salmonella Typhimurium OMP, LPS, and crude extract (CE) and E. coli OMP,
CE, and LPS. Five candidate aptamers with a high level of specificity were identified for further characterization. Serial dilutions of Salmonella Typhimurium pure cultures followed by aptamer capture narrowed down the list to two candidates with low end detection limits at 10-40 organisms. These two candidates, aptamer 33 and aptamer 45, were further analyzed for sensitivity using a magnetic bead based capture protocol using S. Typhimurium cultures and spiked bovine fecal samples. Salmonella was detectable at levels as low as 40 organisms in 1g of fecal sample by Real Time PCR of the DNA isolated from the beads after capture. Competitive binding assays revealed that cross reactivity with the outer membrane proteins of three different enterobacteria, Enterobacter aerogenes, Klebsiella pneumoniae and Shigella flexnerii was abrogated in the presence of S.Typhimurium outer membrane protein. Partially replicated DNase footprint assay reveal that the aptamer binds S. Typhimurium OMP.
Varying affinities to 7 other serovars of the genus Salmonella have been noted. The serovars tested were S. Dublin, S. Enteritidis, S. Fresno, S. Hadar, S. Javiana, S, Moscow, S. Newport. This is the first report where aptamers have been used as high affinity ligands for the detection of bacteria without any pre-enrichment steps.
Impacts
The selected ligands are expected to simplify and improve sensitivity of detection od Salmonella, a leading cause of food bore illnesses in the United States.
Publications
- R. Joshi, V. C. Lopes, K. V. Nagaraja, Lee-Ann Jaykus, J. Schefers, S. Sreevatsan.Application of DNA aptamers as capture molecules to detect Salmonella enterica serovar Typhimurium from complex matrices. Presented at the 2006 Conference for Research workers in Animal Disease Dec 3-5, 2006 Chicago, IL. Abstract Number:95 (food and Environmental Safety Section).
|
Progress 01/01/05 to 12/31/05
Outputs
Methods presently applied to detect and quantitate pathogens such as direct Real Time PCR, particularly in complex matrices such as food, fecal and environmental samples though sensitive, suffer from many drawbacks which do not allow the accurate estimation of pathogen load. The current study is investigating the pre-analytical sample processing using high affinity oligonucleotide ligands (also called aptamers) that bind Salmonellae and Cryptosporidia, in whole cell capture applications to improve assessment pathogen loads. As a first step, aptamers that bind Salmonella Typhimurium are under development. A modified iterative process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is used to enrich Salmonella-specific aptamers. An enriched pool of ligands that reproducibly bind to Salmonella was obtained after seven rounds of counter-SELEX with Salmonella Typhimurium outer membrane protein preparation as the target. E. coli crude outer membrane
preparations and lipopolysaccharides extracts were used in the counter-SELEX in the last few rounds of Counter-SELEX to eliminate potentially cross reacting ligands. The final round of aptamers have been cloned and sequenced. The specificity of the aptamers is being evaluated based on chemiluminiscent gel shift assays with both Salmonella and E. coli outer membrane preparations and their respective lipopolysaccharides preparations. Of the 47 candidates screened, two aptamers have been identified as specific to bind Salmonella with limited cross reactivity to E. coli. Whole cell Salmonella capture using biotinylated aptamers coupled to streptavidin coated magnetic revealed that the low end detection limit was 10 organisms when coupled with a diagnostic invA PCR for Salmonella. These two aptamer candidates are being further tested and optimized for Salmonella capture in complex matrices such as fecal samples from different animal species, food samples and environmental samples. Similar
studies on Cryptosporidium will begin after salmonella specific analysis is completed.
Impacts
The studies underway are expected to improve preanalytical sample processing to enable complete pathogen recovery from complex matrices such as fecal samples, foods, carcass wahes etc. These studies are expected to aid in accurate quantitation of pathogen loads thus contributing to food safety interventions.
Publications
- No publications reported this period
|
|