The application of quantitative-PCR for high throughput screening of novel compounds against cryptosporidium parvum In Vitro and their subsequent IC50

A quantitative-PCR (Q-PCR) method that uses an in vitro culturing system for Cryptosporidium parvum has been developed. This sensitive method allows standardization of an in vitro culturing system and its development for quantitative assessment using PCR. This chapter presents a study in which this system was assessed against an established counting method which is widely used to enumerate parasites, particularly following exposure to anti-parasitic 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 summarized as cumulative effects because of 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 is a consistent and reliable system which offers higher sensitivity and specificity when compared with counting methods as well as providing a vast improvement in sample.