The development of a real-time quantitative-PCR method for characterisation of a Cryptosporidium parvum in vitro culturing system and assessment of drug efficacy

A reliable and sensitive quantitative-PCR (Q-PCR) method using an in vitro culturing system for Cryptosporidium parvum has been developed and characterised. This method allows standardisation of an in vitro culturing system and its development for quantitative assessment using PCR. This system was assessed against an established counting method which is widely used to enumerate parasites, particularly following exposure to antiparasitic compounds. There are several sources of variability inherent in in vitro culturing systems which could result in an inaccurate final amount of DNA being detected per culture well. These can be summarised as cumulative effects due to variability in the in vitro system, and the DNA extraction and quantification method. Analysis of the variability in this in vitro culturing system using Q-PCR indicates that it offers higher sensitivity and specificity when compared with counting methods as well as providing a vast improvement in sample throughput and efficiency. Further to this, we have also determined a method for calculating the inhibitory concentration of anticryptosporidial compounds and present a comparison of this method with a counting method and published data. We conclude that this method of quantification could be used as a substitute for haemocytometer methods, particularly, and also antibody-based techniques.