Developmental stage of strongyle eggs affects the outcome variations of real-time PCR analysis

Strongyle and trichostrongyle parasites are ubiquitous nematodes of grazing livestock. 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 developmental stages can affect the variation of diagnostic test results. This study investigated the influence of developmental stages of strongyle eggs on the variation 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 demineralized 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 examined on an inverted microscope at 100× magnification, where the developmental stages of the eggs were noted, and then 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). SYBR Green Real-Time PCRs were performed in triplicates. Results revealed a statistically significant increase in PCR yield after three days, which was statistically associated with beginning embryonation of the eggs. In conclusion, storage time and developmental stage of strongyle eggs are significant sources of error in studies based on quantitative real-time PCR analysis. This study suggests that for refrigerated storage of more than three days, eggs should be inactivated and preserved for further analysis.