@article{suh_dwivedi_jaykus_2014, title={Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni}, volume={56}, ISSN={["1096-1127"]}, DOI={10.1016/j.lwt.2013.12.012}, abstractNote={A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log10/300 μl C. jejuni cells, one log10 better (lower) than that of IMS-qPCR (2.1 log10 CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log10 CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.}, number={2}, journal={LWT-FOOD SCIENCE AND TECHNOLOGY}, author={Suh, Soo Hwan and Dwivedi, Had P. and Jaykus, Lee-Ann}, year={2014}, month={May}, pages={256–260} }
 @article{suh_dwivedi_choi_jaykus_2014, title={Selection and characterization of DNA aptamers specific for Listeria species}, volume={459}, ISSN={["1096-0309"]}, DOI={10.1016/j.ab.2014.05.006}, abstractNote={Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37 °C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.}, journal={ANALYTICAL BIOCHEMISTRY}, author={Suh, Soo Hwan and Dwivedi, Hari P. and Choi, Soo Jung and Jaykus, Lee-Ann}, year={2014}, month={Aug}, pages={39–45} }
 @article{escudero-abarca_suh_moore_dwivedi_jaykus_2014, title={Selection, Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains}, volume={9}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0106805}, abstractNote={Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.}, number={9}, journal={PLOS ONE}, author={Escudero-Abarca, Blanca I. and Suh, Soo Hwan and Moore, Matthew D. and Dwivedi, Hari P. and Jaykus, Lee-Ann}, year={2014}, month={Sep} }
 @article{dwivedi_smiley_jaykus_2013, title={Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting}, volume={97}, ISSN={["1432-0614"]}, DOI={10.1007/s00253-013-4766-4}, abstractNote={Alternative ligands such as nucleic acid aptamers can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. DNA aptamers demonstrating binding specificity to Salmonella enterica serovar Typhimurium were identified by whole-cell-systematic evolution of ligands by exponential enrichment (SELEX) beginning with a combinatorial library of biotin-labeled single stranded DNA molecules. Aptamer specificity was achieved using whole-cell counter-SELEX against select non-Salmonella genera. Aptamers binding to Salmonella were sorted, cloned, sequenced, and characterized for binding efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively high binding affinity with an apparent dissociation constant (K d value) of 1.73 ± 0.54 μM and was selected for further characterization. Binding exclusivity analysis of S8-7 showed low apparent cross-reactivity with other foodborne bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a ligand for magnetic capture of serially diluted Salmonella Typhimurium cells, followed by downstream detection using qPCR. The lower limit of detection of the aptamer magnetic capture-qPCR assay was 10(2)-10(3) CFU equivalents of Salmonella Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to 12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole cells; (b) the application of flow cytometry for aptamer pool selection, thereby favoring purification of ligands with both high binding affinity and targeting abundant cell surface moieties; and (c) the use of pre-labeled primers that circumvented the need for post-selection ligand labeling. Taken together, this study provides proof-of-concept that biotinylated aptamers selected by whole-cell SELEX can be used in a qPCR-based capture-detection platform for Salmonella Typhimurium.}, number={8}, journal={APPLIED MICROBIOLOGY AND BIOTECHNOLOGY}, author={Dwivedi, Hari P. and Smiley, R. Derike and Jaykus, Lee-Ann}, year={2013}, month={Apr}, pages={3677–3686} }
 @misc{dwivedi_jaykus_2011, title={Detection of pathogens in foods: the current state-of-the-art and future directions}, volume={37}, ISSN={["1549-7828"]}, DOI={10.3109/1040841x.2010.506430}, abstractNote={Over the last fifty years, microbiologists have developed reliable culture-based techniques to detect food borne pathogens. Although these are considered to be the “gold-standard,” they remain cumbersome and time consuming. Despite the advent of rapid detection methods such as ELISA and PCR, it is clear that reduction and/or elimination of cultural enrichment will be essential in the quest for truly real-time detection methods. As such, there is an important role for bacterial concentration and purification from the sample matrix as a step preceding detection, so-called pre-analytical sample processing. This article reviews recent advancements in food borne pathogen detection and discusses future methods with a focus on pre-analytical sample processing, culture independent methods, and biosensors.}, number={1}, journal={CRITICAL REVIEWS IN MICROBIOLOGY}, author={Dwivedi, Hari P. and Jaykus, Lee-Ann}, year={2011}, month={Feb}, pages={40–63} }
 @article{dwivedi_smiley_jaykus_2010, title={Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX}, volume={87}, ISSN={["1432-0614"]}, DOI={10.1007/s00253-010-2728-7}, abstractNote={The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d) value) of 292.8 +/- 53.1 nM with 47.27 +/- 5.58% cells fluorescent (bound) in a 1.48-microM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25-36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1-5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.}, number={6}, journal={APPLIED MICROBIOLOGY AND BIOTECHNOLOGY}, author={Dwivedi, Hari P. and Smiley, R. Derike and Jaykus, Lee-Ann}, year={2010}, month={Aug}, pages={2323–2334} }
 @article{joshi_janagama_dwivedi_kumar_jaykus_schefers_sreevatsan_2009, title={Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars}, volume={23}, ISSN={["0890-8508"]}, DOI={10.1016/j.mcp.2008.10.006}, abstractNote={Sensitive and specific pre-analytical sample processing methods are needed to enhance our ability to detect and quantify food borne pathogens from complex food and environmental samples. In this study, DNA aptamers were selected and evaluated for the capture and detection of Salmonella enterica serovar. Typhimurium. A total of 66 candidate sequences were enriched against S. Typhimurium outer membrane proteins (OMPs) with counter-selection against Escherichia coli OMPs and lipopolysaccharides (LPS). Specificity of the selected aptamers was evaluated by gel-shift analysis against S. Typhimurium OMP. Five Salmonella-specific aptamer candidates were selected for further characterization. A dilution-to-extinction capture protocol using pure cultures of S. Typhimurium further narrowed the field to two candidates (aptamers 33 and 45) which showed low-end detection limits of 10-40CFU. DNase protection assays applied to these aptamers confirmed sequence-specific binding to S. Typhimurium OMP preparations, while South-Western blot analysis combined with mass spectrometry identified putative membrane proteins as targets for aptamer binding. Aptamer 33 was bound to magnetic beads and used for the capture of S. Typhimurium seeded into whole carcass chicken rinse samples, followed by detection using quantitative real-time RT-PCR. In a pull-down assay format, detection limits were 10(1)-10(2)CFU S. Typhimurium/9mL rinsate, while in a recirculation format, detection limits were 10(2)-10(3)CFU/25mL rinsate. Reproducible detection at <10(1)S. typhimurium CFU/g was also achieved in spike-and-recovery experiments using bovine feces. The pull-down analysis using aptamer 33 was validated on 3 naturally infected chicken litter samples confirming their applicability in the field. This study demonstrates the applicability of Salmonella specific aptamers for pre-analytical sample processing as applied to food and environmental sample matrices.}, number={1}, journal={MOLECULAR AND CELLULAR PROBES}, author={Joshi, Raghavendra and Janagama, Harish and Dwivedi, Hari P. and Kumar, T. M. A. Senthil and Jaykus, Lee-Ann and Schefers, Jeremy and Sreevatsan, Srinand}, year={2009}, month={Feb}, pages={20–28} }