Detection and Quantification of Cryptosporidium parvum in Natural Soil Matrices and Leachates Using qPCR
thesisposted on 11.06.2014, 00:00 by Emre Koken
Detection and quantification of Cryptosporidium parvum (C. parvum) oocysts in natural soil matrix and leachates are critical to elucidate the fate and transport of C. parvum in the nature, assess the risk of contamination of water resources and potential threat to public health. Traditional detection and quantification methods are time-consuming, labor-intensive, lack of sensitivity and specificity. Real-Time Quantitative Polymerase Chain Reaction (qPCR) overcomes the limitations in traditional methods and produces sensitive and accurate detections of small numbers of C. parvum. A few qPCR studies were present in the literature for detection and quantification of C. parvum in environmental water samples; however C. parvum in natural soil matrices and leachates have not been researched yet. This research was focused on developing a qPCR protocol for sensitive and specific detection and quantification of C. parvum in natural soil matrices and leachates. The physicochemical parameters—lysis media (TE buffer, Chelex-100), number of thermal shocks (5, 10 or 15) and thawing temperatures (37, 65 or 95oC)—controlling the DNA extraction efficiency were investigated. The oocyst age was found to be the most critical parameter affecting the oocyst disruption and it was found to be less dependent when oocysts were disrupted in TE than in Chelex-100 buffer. Changes in thawing temperature and number of thermal shocks were found more remarkable in aged than in young oocysts and increasing the number of thermal shocks beyond five did not improve the oocyst disruption. The most efficient oocyst disruption method for C. parvum oocysts regardless of their age was then established as 5 thermal shocks with thawing at 65oC in TE buffer. The DNA extraction method was coupled with purification columns to remove PCR inhibitors in environmental samples and additional PCR facilitators—MgCl2 and BSA—were examined to remove residual inhibitors and improve the amplicon yield. The combination of 3 mM MgCl2 and 600 ng/µl BSA yielded highest amplicon yield for both young and aged oocysts. The optimized parameters of DNA extraction and qPCR assay provided very specific and sensitive detections of C. parvum in our study. The minimum detection limit was 0.667 for young and 6.67 for aged C. parvum per PCR reaction and the accuracy of the detections and quantifications were 0.999. The performance of developed protocol was further tested in contrasting soil and leachate samples on the basis of PR values. Mean PR values were calculated as 40±20% Trenton, Greenson and Sparta soil leachates and 43±13% for DI water. Sucrose flotation was determined as a better isolation method than two-phase flotation and it was further experimented for varying concentrations of C. parvum. The lowest and highest PR values were determined as 4.3% and 107.8%, respectively.