Detection, 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

Lombard, Marile (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

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.

AFRIKAANSE OPSOMMING: Die inwendige parasiete Ascaris lumbricoides en Cryptosporidium parvum is hoogs weerstandbiedig teen tradisionele waterbehandelings prosesse en nadelige gesondheids gevolge in individue wat in aanraking kom met die parasiete deur gekontamineerde water, is al aangemeld. Die navorsing uitgevoer in die huidige studie is gekontrakteer deur die East Rand Water Care Company (ERWAT) met die primêre doel om ‘n kwantitatiewe werklike-tyd PKR (qPKR) protokol te optimiseer vir die deteksie en kwantifisering van Cryptosporidium parvum oösiste in drinkwater en omgewingswater, asook die ontwikkeling van ‘n qPKR toets vir die deteksie en kwantifisering van Ascaris eiers in slykmonsters. Die eerste fase van die studie het gefokus op die ontwikkeling van ‘n kwantitatiewe werklike-tyd PKR gekoppel met hoë resolusie smelt kurwe (qPKR-HRM) analise vir die deteksie en kwantifisering van Ascaris lumbricoides eiers in slykmonsters. Verskeie DNS ekstraksie protokolle is met mekaar vergelyk. Na aanleiding van die herhaalbaarheid van die resultate, is die QIAamp Fast DNA Stool Mini kit in samewerking met kraal klop en vries-kook siklusse die mees effektiefste gevind vir die ekstraksie van DNS van A. lumbricoides eiers. Die gebruik van die SensiFAST HRM kit met die Asc1-F/Asc2-R inleier stel (amplifiseer die sitochroom b fragment van A. lumbricoides mtDNS) was suksesvol vir die deteksie van die inwendige parasiet in slykmonsters. Standaard kurwes vir die kwantifisering van A. lumbricoides in die slykmonsters is opgestel deur gebruik te maak van sintetiese geen fragmente (gBlocks Gene Fragments). ‘n Minimum van 1x10-5 ng/μl A. lumbricoides eiers is egter benodig, wat hoёr was as die konsentrasie gevind in die slykmonsters en het dus verdere kwantifisering ingeperk. Die ge-optimiseerde metode is daarna toegepas op slykmonsters van ses verskillende rioolwater behandelings werke vir die deteksie (teenwoordigheid/afwesigheid) van A. lumbricoides. Die qPKR-HRM toets is ook met ‘n kommersiële inleier-peiler gebaseerde qPKR protokol (Genesig kit) vergelyk en die resultate het aangedui dat die molekulêre gebaseerde metodes die potensiaal het om die mikroskopie metode wat tans deur ERWAT gebruik word vir die deteksie van Ascaris sp. te vervang. Die Genesig protokol het ook die bygevoegde voordeel gehad dat die hoeveelheid eiers in die monsters gekwantifiseer kon word met die standaardkurwe wat deel uit maak van die protokol reagense. Die koste- en tydeffektiwiteit van elke metode is bepaal en vergelyk. Die qPKR-HRM toets en die Genesig protokol het soortgelyke omkeertye opgelewer van ±7 ure in vergelyking met die ERWAT mikroskopie metode van ±50 ure. Daarby het koste vergelykings gewys dat die qPKR-HRM toets die mees koste-effektiewe (R684.75) metode was vir die deteksie van A. lumbricoides in slykmonsters, gevolg deur die ERWAT mikroskopie metode (R706.86) en dan die Genesig protokol (R900.36). In die tweede fase van die studie is die qPKR-HRM toets geoptimiseer vir die deteksie en kwantifisering van Cryptosporidium parvum oösiste in watermonsters. Verskeie DNS ekstarksie protokolle is met mekaar vergelyk en resultate het gewys dat die QIAamp Fast DNA Stool Mini kit (geen modifikasies) die mees effektiefste was vir die ekstraksie van DNS van C. parvum oösiste vanuit stoelgangmonsters. Die gebruik van die SensiFAST HRM kit met die COWP-F/COWP-R inleier stel (amplifiseer die C. parvum oösist wand proteïen) was suksesvol vir die deteksie van die inwendige parasiet. Standaard kurwes vir die kwantifisering van C. parvum in die monsters is opgestel deur gebruik te maak van sintetiese geen fragmente gebaseer op die C. parvum oösist wand proteïen. Weereens kon die standaard kurwe slegs C. parvum DNS kwantifiseer tot by 1x10-5 ng/μl wat hoёr was as die konsentrasie van C. parvum DNS in die stoelgangmonsters en die standaard kurwes kon nie verder gebruik word vir kwantifisering nie. Die ge-optimiseerde metode is daarna toegepas vir die deteksie (teenwoordigheid/afwesigheid) van C. parvum oösiste in drinkwater met bygevoegde stoelgangmonsters en daar is bevind dat die monster limiet van deteksie van die qPKRHRM toets minder as 1 oösist/ml was. Die qPKR-HRM toets is ook toegepas op oppervlakwater monsters geneem op en afstroom van ‘n rioolwater behandelings werke vir die deteksie van Cryptosporidium sp. en is vergelyk met ‘n kommersiële inleier-peiler gebaseerde qPKR protokol (Genesig kit). Die qPKR-HRM toets was meer sensitief vir die deteksie van C. parvum DNS, alhoewel die kommersiële protokol die kwantifisering van die hoeveelheid oösiste in een van die monsters bewerkstellig het. Terwyl soortgelyke omkeer tye van ±7 ure vir beide metodes gevind is, het koste vergelykings gewys dat die qPKR-HRM toets meer koste effektief was (R578.96) as die Genesig kit (R794.57) vir monster analise.

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