The developmental stage of strongyle eggs affects the outcome of real-time PCR analysis

Several molecular diagnostic tests are based upon measuring and quantifying DNA obtained from parasite eggs. It is well-known that such eggs undergo development during storage, but it remains unknown to which extent the stage of development can affect the diagnostic test result. This project investigated whether the developmental stage of strongyle eggs affects real-time polymerase chain reaction (PCR) results. Mixed species strongyle eggs were obtained from the faeces of a naturally infected horse. Eggs were isolated and placed in microtiter plates with demineralised water. A total of 25 wells containing 100 eggs each were set up and kept refrigerated for up to five days. Once daily, five wells were microscopied on an inverted microscope, the developmental stages of the eggs were noted, and the eggs harvested for DNA extraction. The protocol was repeated three times. Genomic DNA was extracted using a commercial kit previously validated for strongyle type eggs. PCR reactions were performed with a primer set specific for the ribosomal DNA region for all strongyle type parasites (NC1, NC2). PCRs were performed in triplicates using SYBR Green as fluorescent dye. PCR results were registered as cycle of threshold (Ct) values. Statistical analysis revealed significant differences between days. Results illustrated a significant increase in PCR yield after three days, which was associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle egg are significant sources of error in studies
based on quantitative real-time PCR analysis performed. For storage more than three days, eggs should be killed and kept on ethanol for further analysis.