Browsing by Author "Lombard, Marile"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemDetection, identification and quantitation of Cryptosporidium parvum in water samples and Ascaris lumbricoides in sludge samples using real-time Polymerase Chain Reaction coupled with the high-resolution melt curve assay(Stellenbosch : Stellenbosch University, 2016-03) Lombard, Marile; Khan, Wesaal; Wolfaardt, Gideon M.; Van Blerk, Nico; Stellenbosch University. Faculty of Science Dept. of Microbiology.ENGLISH ABSTRACT: The intestinal parasites Ascaris lumbricoides and Cryptosporidium parvum are highly resistant to traditional water treatment processes and adverse health effects in individuals that come into contact with these parasites through contaminated water, have been reported. The research conducted in the current study was contracted by the East Rand Water Care Company (ERWAT) and the primary aim was to optimise a quantitative real-time PCR (qPCR) protocol for the detection and quantitation of Cryptosporidium oocysts in drinking water and environmental surface waters and develop a qPCR assay for the detection and quantitation of Ascaris eggs in sludge samples. The first phase of the study focussed on developing and optimising a quantitative real-time PCR coupled with high-resolution melt curve (qPCR-HRM) assay for the detection and quantitation of Ascaris lumbricoides eggs in sewage sludge samples. Various DNA extraction protocols were compared and based on the reproducibility of the results, the QIAamp Fast DNA Stool Mini kit in conjunction with bead beating and freeze-boil cycles, was the most effective for the extraction of DNA from A. lumbricoides eggs. The use of the SensiFAST HRM kit with the Asc1-F/Asc2-R primer set (amplifying the cytochrome b fragment of A. lumbricoides mtDNA) also proved successful for the detection of the intestinal parasite in sludge samples. Standard curves for the quantitation of A. lumbricoides in the samples were also constructed using synthetic gene fragments (gBlocks Gene Fragments). However, a standard curve with dilutions up to 1x10-5 ng/μl could only be constructed and as the concentration detected in the sludge samples was lower, further quantitation was limited. The optimised method was then applied to sludge collected from six different wastewater treatment plants for the detection (presence/absence) of A. lumbricoides. The qPCR-HRM assay was also compared to a commercial primer-probe based qPCR detection kit (Genesig kit) and the results indicated that the molecular based methods have the potential to replace the currently employed microscopy method for the detection of Ascaris sp. The Genesig kit had the added advantage of quantifying the number of eggs present in the samples. The cost- and time-effectiveness of each method was calculated and compared. The qPCR-HRM assay and Genesig kits yielded similar turnaround times of ±7 hours compared to the microscopy method that yielded a turnaround time of ±50 hours in total. In addition, cost comparisons showed that the qPCR-HRM assay was the most cost-effective (R684.75) method for the detection of A. lumbricoides in sludge samples, followed by the ERWAT microscopy method (R706.86) and then the Genesig kit (R900.36). In the second phase of the study a qPCR-HRM assay for the detection and quantitation of Cryptosporidium parvum oocysts in water samples was optimised. Various DNA extraction protocols were compared and results showed that the QIAamp Fast DNA Stool Mini kit (no modifications) was the most effective for the extraction of DNA from C. parvum oocysts. The use of the SensiFAST HRM kit with the COWP-F/COWP-R primer set (amplifying the C. parvum oocysts wall protein) proved successful for the detection of the intestinal parasite. Standard curves intended for the quantitation of C. parvum in the samples were constructed using synthetic gBlocks Gene Fragments based on the C. parvum oocyst wall protein. Once again the standard curves could only quantify the C. parvum DNA up to 1x10-5 ng/μl, which was below the concentration of C. parvum DNA in the faecal samples and the standard curves could not be utilised for further quantitation. The optimised method was then applied for the detection (presence/absence) of C. parvum oocysts in drinking water spiked with faecal samples and it was determined that the sample limit of detection of the qPCR-HRM assay was less than 1 oocyst/ml. The qPCR-HRM assay was also applied to surface water samples collected up and downstream of a wastewater treatment plant for the detection of Cryptosporidium sp. and was compared to a commercial primerprobe based qPCR detection kit (Genesig kit). The qPCR-HRM assay was more sensitive in detecting C. parvum DNA, although the commercial kit was able to quantify the number of Cryptosporidium sp. oocysts in one of the samples. While similar turnaround times of ±7 hours were obtained for both methods, cost comparisons showed that the qPCR-HRM assay was more cost effective (R578.96) than the Genesig kit (R794.57) for sample analysis.