Browsing by Subject "PCR"
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Item A comparison of diagnostic techniques for detecting salmonella spp in equine fecal samples using culture methods, gel-based pcr, and real-time pcr assays(Texas A&M University, 2007-09-17) Smith, Shelle AnnSalmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.Item Analysis of the Microbial Community Along Three Sites of the Rio Grande by Denaturing Gradient Gel Electrophoresis(Texas A&M International University, 2015-06) Castro, Rebeca Imelda; Mendez, Monica O.ABSTRACT Analysis of the Microbial Community of Three Sites Along the Rio Grande by Denaturing Gradient Gel Electrophoresis (May 2014) Rebeca Imelda Castro, B. A., Texas A & M International University; Chair of Committee: Dr. Monica Mendez In the last decades, the increase in population growth and differences in enforcement regulations on both sides of the Rio Grande may have affected the bacterial composition as a result of effluent discharges, industrial waste, raw sewage outfalls, and agricultural runoff. The purpose of this study was to compare structural differences in the microbial communities of the Rio Grande-Rio Bravo waters based on Denaturing Gradient Gel Electrophoresis (DGGE) analysis. Three sites were selected based on proximity to anthropogenic influences with the highest level to the lowest level of potential human impact (from the site furthest downstream to upstream) as follows: Site 1-Zacate Creek Area located downstream of the Zacate Waste Water Treatment Plant; Site 2-Bridge I, located upstream of the U.S.-Mexico International Bridge I; and Site 3- Jefferson Water Treatment Plant Intake located upstream of Site 2-Bridge I. In order to assess environmental factors potentially affecting the microbial composition at these sites, physiochemical parameters were determined such as: water temperature, conductivity, pH, depth, chlorophyll a, optical dissolved oxygen, total nitrogen and total phosphorous. The microbial community was analyzed using heterotrophic plate counts on R2A agar and by PCR amplification of the 16S rRNA gene followed by DGGE analysis. Although water quality parameters were similar, differences between the sites were noted for the microbial communities. Heterotrophic plate counts were significantly highest (p = 0.0006) at Site1-ZCA and the lowest at Site 3-JWTP. Based on DGGE analysis, similar trends were observed with the OTU richness which was highest for Site 1-ZCA (28 OTUs), which had six unique OTUs, and lowest for Site3-JWTP. Possible influences could be eutrophic conditions from a build-up of nutrients including dissolved organic matter, salts or other inorganic contaminants, most likely from sewage and waste water discharges nearest Sites 1 and 2. Results suggest that general water quality parameters may not be sufficient in determining potential anthropogenic impacts on the microbial community, and that other factors not assessed in this study could be impacting the bacterial richness and diversity of the Rio Grande.Item Assessment of a Proposed Method of Trypanosoma cruzi Detection in Frozen Mammalian Tissues from Natural History Collections in TexasSkinner, Kalin Michelle; Negovetich, Nicholas J; Strenth, Ned E; Ammerman, Loren K; Hung, You-jou; Fohn, Laurel EThis study examined a proposed method of detecting the presence of Trypanosoma cruzi in tissue samples of mammalian museum specimens proposed by Pinto et al. (2010). Pinto et al (2010) detected the presence of T. cruzi in 42 out of 159 Neotoma micropus from the Chaparral Wildlife Management Area. This study re-examined the 42 T. cruzi-positive tissue samples used in Pinto’s study and failed to detect a single positive for T. cruzi. In addition, 50 samples of 16 species of mammals from Concho and Val Verde counties from the Angelo State Natural History Collection were tested for the presence of T. cruzi. None of these tissue samples tested positive for presence of the parasite. The results of this current study do not support the use of frozen tissues from museum collections for the detection of the parasite T. cruzi from a known endemic geographical region. Several proposals are presented in an effort to explain the discrepancy in the results of these two studies.Item Comparison of Three Techniques for Detecting Enterotoxin A (SEA) in Clinically Relevant Staphylococcus aureus StrainsHarrison, Cari Riccay; Jones, Crosby W; Ammerman, Loren K; Guardiola, Amaris R; Keith, Susan EThirty-three clinical strains of Staphylococcus aureus were screened for the production of staphylococcal enterotoxin A (SEA) protein and/or its gene using three techniques, reversed passive latex agglutination (RPLA), Ouchterlony double diffusion (ODD), and polymerase chain reaction (PCR). Of the isolates, two produced detectable levels of SEA (6%), while 22 (64.7%) harbored the gene for SEA. These results indicate that clinical S. aureus isolates commonly have the potential of producing SEA. ODD testing revealed promise for using it to detect prolific producers of enterotoxins. RPLA was shown to detect enterotoxins with specificity and accuracy. PCR revealed that although clinical strains frequently have the sea gene, they often do not produce SEA. Further studies should examine the presence of other staphylococcal enterotoxin genes and products. Growth conditions should also be evaluated to determine the ideal environments for enterotoxin production.Item Design and characterization of convective thermal cyclers for high-speed DNA analysis(2009-05-15) Agrawal, NitinAn ideal polymerase chain reaction (PCR) system should be capable of rapidly amplifying a wide range of targets in both single and multiplex formats. Unfortunately, the timescales and complexities involved in many existing technologies impose significant limitations on achievable throughput. Buoyancy driven PCR is emerging as a simplified version of thermally driven bio-analysis systems. Here, we demonstrate a simplified convectively driven thermocycler capable of performing single and multiplex PCR for amplicons ranging from 191 bp to 1.3 kb within 10 to 50 minutes using 10 to 25 ?L reaction volumes. By positioning two independent thermoelectric heating elements along the perimeter of a flow loop reactor constructed using ordinary plastic tubing, a buoyancy-driven flow is established that continuously circulates reagents through temperature zones associated with the PCR process. Unlike conventional benchtop thermocyclers, this arrangement allows reactions to be performed without the need for dynamic temperature control of inactive hardware components while maintaining comparable product yields and requiring no modifications to standard PCR protocols. We also provide a general correlation that can be applied to design reactor geometries satisfying virtually any combination of reagent volume and cycling time. In addition to offering an attractive combination of cost and performance, this system is readily adaptable for portable battery powered operation, making it feasible to perform PCRbased assays in a broader array of settings.Item Diversity of Low Chill Peaches (Prunus persica) from Asia, Brazil, Europe and the USA(2011-08-08) Anderson, Natalie A.One hundred fifty-five peach (Prunus persica) cultivars, from Asia, Brazil, Europe, and the USA, were examined using eleven Simple Sequence Repeats (SSRs) to study the genetic relationships among low chill as compared to high chill peach germplasm. Data was analyzed by NTSYSpc to form a similarity matrix using Nei and Li?s Dice similarity coefficient. This similarity matrix was then subjected to a cluster analysis and a dendrogram was constructed using the UPGMA (Unweighted Pair-Group Method, Arithmetic Mean) method. A wide range of diversity was detected, from 0.33 coefficient of similarity amongst the Thai peaches to 0.97 between two Brazilian peaches. The most distant clusters were the low chill peaches from Thailand and Taiwan and the local cultivars (both fruit and ornamental types) from China. Among the improved germplasm, there were distinct clusters for the Chinese/Japanese cultivars, three clusters for the Brazilian cultivars and one for the cultivars from the USA and Europe. The Brazilian materials clustered according to breeding programs in S?o Paulo and Pelotas reflecting the different sets of local cultivars used in the breeding efforts. The largest group investigated was the European/USA peaches. This group subdivided into three distinct clusters, with a general clustering of the low chill germplasm. The low chill accessions from Asia were genetically distant from the improved low chill peaches from the USA or Brazil. The low chill peaches from the Americas were more closely related to the high chill peaches developed in the USA and China/Japan due to the introgression of this germplasm into a low chill background.Item Molecular characterization of cation-coupled transporters: the H+-coupled Mg2+-citrate transporter, CitM, and the Na+/sulfate cotransporter, hNaSi-1(2003-01-28) Hongyan Li; Ana M. Pajor; Steven C. King; Steven A. Weinman; Luis Reuss; Joel P. GallagherIn this dissertation, two cation-coupled transporters were characterized at the molecular level. The CitM transporter from Bacillus subtilis was functionally expressed and characterized in E.coli cells. The human NaSi-1 transporter (hNaSi-1) and mutants were functionally expressed in Xenopus oocytes. Antibodies against hNaSi-1 were used to investigate tissue distribution and N-glycosylation. The roles of two conserved serine residues in the transport function of hNaSi-1 were investigated using site-directed mutagenesis and radiotracer assay. \r\n\r\n CitM belongs to a distinct gene family of secondary active transporters that includes the homologous citrate transporter CitH. In this dissertation, the Km of CitM for the complex of Mg2+-citrate was about 300 mM in the presence of saturating Mg2+ concentrations. CitM has a high substrate specificity for citrate. Other tested di- and tricarboxylic acids did not significantly inhibit citrate uptakes in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The transport was inhibited in more alkaline but not in acidic transport buffer and also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin, suggesting a proton-coupled transport. Valinomycin did not affect the uptake by CitM, supporting an electroneutral transport model in which one proton is coupled to the uptake of one complex of (Mg2+-citrate)1-. \r\n\r\nThe low affinity Na+/sulfate cotransporter, hNaSi-1, belongs to a specific gene family of Na+-coupled transporters that includes the high affinity hSUT-1 and the Na+-coupled dicarboxylate (NaDC) transporters. Antibodies directed against a peptide of hNaSi-1 recognized the native protein in renal membranes as well as the recombinant protein expressed in Xenopus oocytes. There is a single N-glycosylation site, Asn-591, located at the extracellular C-terminus in hNaSi-1. Site-directed mutagenesis studies of Ser-260, Ser-288 and the surrounding amino acid residues of hNaSi-1 suggested that these residues are functionally required for hNaSi-1. MTSET inhibition on sulfate uptakes by the four mutants surrounding Ser-260, T257C, T259C, T261C and L263C, was dependent on the cation and substrate used. Since the presence of sodium and sulfate triggers conformational changes during the transport cycle of hNaSi-1, the cation and substrate dependence of MTSET inhibition suggest that these four substituted cysteines move during the transport cycle. Since the four mutated residues are located in TMD-5, this transmembrane domain is also likely to participate in the conformational movement during the transport cycle of hNaSi-1. \r\nItem Prevalence of Salmonella sp. in domestic cats in an animal shelter and the comparison of culture and polymerase chain reaction techniques as diagnostic tools(Texas A&M University, 2004-11-15) Lee, Melinda J.Previous studies on the prevalence of Salmonella in cats have used a variety of culture methods producing a variety of results, but none have been compared to PCR. Using a double enrichment protocol developed at Texas Veterinary Medical Diagnostic Laboratory the prevalence of Salmonella in shelter cat feces was determined in this current study. The culture protocol used included Xylose Lysine Tergitol 4 (XLT4) and MacConkey (MAC) agars with a primary enrichment in Tetrathionate broth (TTH) with iodine and a secondary enrichment in Rappoport-Vassilaidis R10 broth (RV). This study further modified an equine PCR technique and demonstrated its successful use in cats. When comparing the results of the two protocols, PCR and culture, it was found that the procedures are equally adequate at detecting the presence of Salmonella in cat feces. This study further confirmed that Salmonella is a potential hazard for families who adopt shelter cats.Item The Mystery of the Chaetognatha: A Molecular Phylogenetic Approach Using Pelagic Chaetognath Species on Pelican Island, Galveston, Texas(2011-02-22) Towers, Leah NicoleThe phylum Chaetognatha is a mysterious group of organisms that has eluded scientists for more than a century because of their unique morphology and developmental characteristics, i.e. protostome (mouth develops from blastopore; e.g. mollusks, annelids, arthropods) versus deuterostome (anus develops from blastopore; e.g. echinoderms and chordates) offer few clues to their evolutionary origins. Some early morphological studies argued that chaetognaths were derived mollusks or nematodes according to gross ultrastructural data, while other studies focused on the coelomic cavity. 33 Although 18S rRNA is widely used in molecular phylogeny studies, it has limits such as long- branch chain attractions and a slow rate of evolutionary change. Long-branch chain attractions are a phenomenon in phylogenetic analyses when rapidly evolving lineages are inferred to be closely related, regardless of their true evolutionary relationships. Hence other genes are used in this study to complement the 18S rRNA such as the cytochrome oxidase genes. The cytochrome oxidase genes are highly conserved throughout all eukaryotic organisms and they are less ambiguous to align as compared to the ribosomal genes, making them better phylogenetic markers as compared to the 18S rRNA gene. This study focuses on using a molecular approach (ARDRA, PCR, phylogenetic tree reconstruction) to determine the phylogeny of pelagic chaetognaths found on Pelican Island, Galveston, Texas. 18S rRNA, Cytochrome Oxidase I and Cytochrome Oxidase II genes were used to help decipher the phylogeny of this group. All analyzed genes in this study (18S rRNA, COI, and COII) grouped the Pelican Island chaetognaths with the protostomes. The maximum parsimony bootstrap tree for the 18S rRNA gene, grouped the samples closest to the arthropods (protostome). For the COI and COII genes, the minimum evolution bootstrap tree grouped the 8 collected samples more closely to two other protostome phyla: the mollusks and annelids (COI) while bootstrapping with the COII grouped the samples with the nematodes (with >66 percent bootstrap). My findings are significant because they reveal phylogenetic results of a protostome lineage for the Chaetognatha using 3 genes, one of which (COII) has not been greatly studied for the Chaetognatha.