M.Sc. / Mnandi and Astrid are two commercially important potato cultivars in South Africa. These two cultivars are closely related and are morphological virtually identical. It is, however, necessary to be able to distinguish between these two cultivars, because each of these cultivars has certain desirable characteristics. It was decided to use DNA markers, since DNA markers are not influenced by the environment and the polymerase chain reaction (PCR) based DNA markers are relatively easy, cheap and fast. It was decided to develop a sequenced characterized amplified region (SCAR) due to the problems with the reproducibility of random amplified polymorphic DNA (RAPDs). SCARs are derived from RAPD fragments by using the sequence of a RAPD derived fragment to design a set of new longer primers (usually 20-24mer) which are less sensitive to PCR conditions. Ten commercial potato cultivars (Astrid, Mnandi. BP,, Buffelspoort, VanderPlank, Up-to-Date, Hoevelder, Hertha, Pimpernel and Agria) were used in this study. Commercially available RAPD primers (102) were evaluated to seek a polymorphism unique to either Mnandi or Astrid. Thirtyseven polymorphisms between Astrid and Mnandi were identified but only three were unique. The polymorphism obtained with OPH-15 was however, not reproducible. The polymorphisms obtained with UBC 509 and 582, corresponding to the presence in Mnandi of a 300 and 900 by fragment respectively, were reproducible. These two fragments, UBC 509 3" and UBC 582900, were cloned into the pMosBlue TA cloning vector and sequenced. The identity if the inserts in the recombinant plasmids were verified with PCR and Southern blotting. The sequences were used to develop two sets of SCAR primers, SCAR UBC 509 3" and SCAR UBC 582900 . The two SCAR primer pairs were then used in PCR reactions. The SCAR UBC 509 300 primer pair amplified a fragment of 230 by in both Astrid and Mnandi and a fragment of 260 by in Mnandi. The polymorphism is thus retained and SCAR UBC 509 3" can be used to distinguish between Astrid and Mnandi. The SCAR UBC 582' primer pair amplify a fragment of 500 by in both Astrid and Mnandi as well as some other longer fragments. It was not possible to regain a polymorphism by either elevating the annealing temperature or by digesting the amplification products with restriction enzymes. SCAR UBC 582' could thus not be used to distinguish between Astrid and Mnandi.

M.Sc. / A common feature of multi gene families is that their members are expressed in different ways in response to environmental and developmental signals. In the present study the expression of a CHS multi gene family in bean (Phase°lus vulgari.), was studied. using a RT-PCR technique that focuses on the 3' divergent regions of the isogenes. Tissue-specific expression in roots, stems, leaves and the flowers of Phaseolus vulgaris, as well as in callus tissue, were investigated. Patterns and levels of gene expression were investigated after treatment with different elicitors as well as light. In most cases time and concentration studies were performed. The four CHS transcripts (CHS4. CHS1. CHS17 and CHS14) showed tissue-specific expression. The four CHS transcripts were differently expressed in the seven organs investigated: and different levels of activity were observed. The highest level of transcript expression for CHS14 and CHS4 could be observed in the roots, whereas relatively low levels were obtained in the leaves. stems and flowers of the green as well as etiolated seedlings. Higher levels of CHS were found in flower buds. High levels of all four transcripts were also found in callus. Elicitor treatment with structurally diverse abiotic agents showed induction of all four CHS mRNA transcripts. Concentration studies revealed high levels of CHS transcript levels. Elicitation with different concentrations of the elicitors: glutathione. mercuric chloride and sodium salicylate showed high levels of the CHS transcripts after exposure of 6 h to the different elicitation agents. The transcript levels increased significantly to levels above those observed in untreated (control) plants. The CHS transcripts showed higher levels of induction after elicitation with mercuric chloride (1 mM) relative to treatment with sodium salicvlate (10 rnM), suggesting differential regulation at the transcriptional level. The expression patterns observed with glutathione were very similarly to those induced by mercuric chloride. The kinetics of induction of all CHS transcripts. except for CHS1 were low at 2 and at 8 h postelicitation and maximal levels of transcript. although transiently induced, could be observed at 4 - 6 h. The use of 4 mM mercuric chloride did not give any induction. most probably because it was a lethal concentration. Etiolated and green bean seedlings, exposed to UV light. showed expression of all four CHS transcripts. In green leaves no significant differences in the induction kinetics between the different chs genes were observed. Three of the transcripts (CHS4. CHSI7. CHS14) accumulated rapidly (within ca. 3h). reaching a maximum after 6 h of irradiation. followed by a decline. CHS4 revealed a 18.2 fold induction. CHSI7 showed a 4.8 fold increase and CHS14 a 4 fold increase after 6 h of illumination in green leaves. In contrast. CHS1 showed a delay ed response which was still observable after 15 h. It was also demonstrated that CHS transcripts accumulated rapidly but transiently. Following illumination of etiolated leaves with white light. except for CHS I. CHS17 and CHS4 showed similar expression levels and patterns. with maximal induction at 1,5 h after white light exposure, whereas maximal induction for CHS14 was at 2 h. At 2.5 Ii the levels for all three transcripts dropped to preinduction levels. It is therefore evident that CHS is a key metabolic control point in the phenylpropanoid pathway leading specifically to isollavonoid biosynthesis. The results strongly suggest that the activation of plant defence genes are regulated in a tissue-specific manner and that induction by different elicitor-active agents. may be regulated by different. But convergent signal transduction regulatory networks.

No description available.

The etiological agent of rabies encephalitis belongs to the genus Lyssavirus in the Rhabdoviridae family. Lyssaviruses are negative sense, single stranded RNA viruses and cause an estimated 55 000 human deaths per year with 44% of these deaths occurring in Africa (WHO, 2005). With intense research effort and increased sequence information it is becoming evident that the Lyssavirus genus is much more diverse than initially thought and therefore diagnostic methods need to be modified accordingly. The African continent sustains a diverse variety of lyssaviruses, however, most countries in Africa do not have active surveillance or necessary diagnostic tools and therefore rabies-related lyssaviruses are underreported. Previous studies have indicated that real-time PCR has improved sensitivity and rapidity over conventional molecular diagnostic methods with the added advantage of allowing accurate estimations of viral load in a wide variety of samples. Several realtime PCR assays have been developed; however, none were specifically aimed at detection of lyssaviruses present on the African continent. This study was therefore aimed at evaluating certain molecular diagnostic methods for the detection of African lyssaviruses. Furthermore, the application of real-time PCR for various fields in lyssavirus research i.e. diagnostics, surveillance and pathogenicity studies were evaluated. This study revealed two different hemi-nested PCR assays capable of detecting representatives of African lyssaviruses. A real-time PCR was developed that was successful for the detection of African lyssaviruses. In addition, a quantitative assay and internal control was successfully employed for confirming ante-mortem human rabies diagnosis as well as post-mortem animal rabies diagnosis in formalin fixed brain material. As such the real-time PCR assay developed in this study could therefore be routinely used for ante-mortem diagnosis and as a confirmatory test for post-mortem diagnosis. The ability of this assay to detect and quantify all currently known African lyssaviruses not only offers improved surveillance capacity, but offers unique potential as a sensitive tool to track virus movement in pathogenicity studies. These aspects are important in our search for a better understanding of the complex epidemiological and viral characteristics of African lyssaviruses. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

>Magister Scientiae - MSc / Insertion-deletion polymorphisms (indels) have been underutilized in forensic identification of individuals in comparison with single nucleotide polymorphisms (SNPs) and short tandem repeat (STRs) systems. The use of indels for the purpose of human identification is more advantageous than previously used methods as it combines desirable characteristics of both the SNPs and STRs i.e. low costs and simplistic typing methods as well as indels having small amplicons size, making them suitable for genotyping highly degraded DNA. Currently there is only one commercial kit available for the forensic community, the Investigator® DIPlex kit (Qiagen), which cover a total of 30 indel loci distributed over 19 autosomal chromosomes. The objective of this study was to evaluate the Qiagen Investigator® DIPplex kit for forensic application in South Africa. The kit‘s performance was evaluated by comparing different extraction methods; sensitivity, robustness and reproducibility were evaluated and forensic parameters (match probability, power of discrimination, polymorphism information content, power of exclusion and typical paternity index) were estimated based on population data generated from five South African populations (Afrikaner, Mixed Ancestry, Indian-Asian, Xhosa and Zulu). Population comparisons were performed using Fst-analysis, factorial component analysis as well as phylogenetic tree construction. DNA was extracted from buccal swabs and whole blood collected from a total of 512 individuals from the five South African population groups and genotyped using the Qiagen Investigator® DIPplex kit. Sanger DNA sequencing and sequence alignments confirmed the presence of a null allele at locus HLD97 which was present in high frequency in the Xhosa and Zulu populations. This observation was made in 14 individuals belonging to the Xhosa and Zulu populations. Null allele frequencies in all five South African populations were also estimated. Null alleles were estimated for all loci using analytical methods i.e. Charkraborty null allele estimator, Brookfield null allele estimators 1 and 2 and ML-NullFreq software program. The kit performed well in the laboratory, not requiring any additional reagents or instrumentation and successfully generating profiles with input DNA amounts as low as 0.2 ng/μL. Although well suited for forensic application, the Qiagen Investigator® DIPplex kit showed some drawbacks with regards to application on South African populations. The presence of a null allele at the HLD97 locus as well as indication of population substructure affects allele frequency estimates for the South African populations. Correction for population substructure as present within the South African populations should be considered using FST analysis and it is recommended that the HLD97 locus should be excluded from any kinship analysis performed on South African populations.

Milk quality continues to be a topic of intense debate in the dairy industry, medical and public health communities. Production of maximum quantities of high-quality milk is an important goal of every dairy operation. High-quality milk must contain a low number of somatic cells and low bacteria count, and must be free of human pathogens and antibiotic residues. The objective of this study was to determine the prevalence of E. coli in unpasteurized milk recovered from Middledrift and Fort Hare dairy. In this study 400 milk samples were collected from two commercial farms (Middledrift and Fort Hare) in the Eastern Cape, South Africa, 200 raw milk samples from each farm. Samples were cultured on violet red bile mug-agar (VRB-MUG Agar) and incubated at 37ºC for 24 hours and preliminary identified by Gram stain and catalase test. Isolates that were Gram negative and catalase positive were screened for a marker of E. coli uidA gene using PCR assays. Middledrift dairy farm had 50 (25%) E. coli isolated from raw milk and Fort Hare farm showed 37 (18.5%) E. coli present in the milk samples. The presence of E. coli found in the milk samples points to the fact that fecal contamination was unavoidable and traditional practices are likely to contribute to the contamination of the milk and proliferation of the microorganisms.

Presented diploma thesis is focused on use of DGGE to analysis and identification of selected microorganisms. PCR-DGGE is a method that allows direct characterization of the microbial community in the natural environment without necessity of cultivation. A literature review is devoted to the principle of the method, current applications and its limitations too. In experimental part microbial DNA was isolated and used as a template for PCR reaction. Microbial DNA was then amplified using the universal eukaryotic primers that target the D1/D2 domain of the 26S subunit of ribosomal DNA. To improve specificity and sensitivity of detection nested PCR was chosen using outer and inner primer pairs. Generated amplicons (250 bp) were consequently separated by DGGE. The analysis of selected microorganisms by DGGE technique was performed after optimization of electrophoresis conditions (in particular the denaturing gradient extent and separation time). Despite the optimization, mutual differentiation among individual yeast strains was not possible since each reference strain was represented by several bands in the same positions. In conclusion DGGE profile obtained from wine musts is discussed. Present bands suggest the major presence of non-Saccharomyces yeasts, yeast-like strain A. pullulans is present in the minority and Saccharomyces yeasts are probably present too. The technique remains open for further optimization, particularly as regards the conditions of polymerase chain reaction.

Pertussis is an infectious disease caused by the Gram-negative bacterium Bordetella pertussis. In Peru, actual public health programs indicate that vaccination against B. pertussis must be mandatory and generalized, besides all detected cases must be reported. The objective of this study was to determine the prevalence of B. pertussis among children under five years of age with a presumptive diagnosis of whopping cough in Cajamarca, a region located in northern Peru. / Background and Objectives: Pertussis is an infectious disease caused by the Gram-negative bacterium Bordetella pertussis. In Peru, actual public health programs indicate that vaccination against B. pertussis must be mandatory and generalized, be-sides all detected cases must be reported. The objective of this study was to determine the prevalence of B. pertussis among children under five years of age with a presumptive diagnosis of whopping cough in Cajamarca, a region located in northern Peru. Materials and Methods: The population of this cross-sectional study were children under 5 years old hospitalized as presumptive cases of pertussis during December 2017 to December 2018. The nasopharyngeal samples were analyzed by real-time PCR for the detection of B. pertussis. Results: B. pertussis was identified as PCR + in 42.3% of our sample (33/78). The clinical presentation that was observed most frequently includes paroxysmal coughing (97%), difficulty breathing (69.7%), cyanosis (72.7%) and post-tussive em-esis (60.6%). Additionally, pneumonia was the most observed complication (33.3%). Four of the patients with PCR+ for B. pertussis presented only lymphocytosis, five only leukocytosis, two patients with decreased leukocytosis and lymphocytes and only one patient with leukopenia and relative lymphocytosis. There was a percentage of 84.8% of unvaccinated children in the PCR+ group. Finally, the mother was the most frequent symptom carrier (18.2%). Conclusion: In conclusion, in the studied population there is a high rate of PCR+ cases for B. pertussis. Laboratory values may show leukopenia or lymphopenia in patients with pertussis. It is necessary to use appropriate laboratory diagnostic tests in all infants with respiratory symptoms for B. pertussis. Since, the clinical diagnosis overestimates the diagnosis of pertussis. / Revisión por pares

Therapy of acute leukemia may be improved by a more accurate assessment of the effects of treatment on tumor burden and by anticipating relapse with greater precision. The sensitivity limit of assessing residual disease by morphology is usually 5%. Several alternative approaches are available to study minimal residual disease, defined as the presence of leukemic cells not detectable by morphology. These include studies of chromosomal abnormalities by conventional karyotyping, flow cytometry, in situ hybridization and polymerase chain reaction (PCR), investigation of gene rearrangements by Southern blotting and PCR, and immunological methods. Some of these techniques enable the detection of 1 leukemic cells among 10 000 or more normal cells. In the following, the advantages and limitations of sensitive methods for detecting small numbers of leukemic cells are reviewed. The rationale for monitoring residual disease in acute leukemia and the initial results of studies correlating minimal residual disease and clinical outcome are discussed.

No description available.