Pre-Analytical Cleanup of Feline Feces Improves DNA Extract Quality, Reduces Post-Extraction PCR Inhibition, and Enhances Molecular Detectability of Intestinal Protozoa.

Feline feces represent a difficult matrix for molecular diagnostics because stool-derived inhibitors and variable sample composition may compromise DNA recovery and downstream PCR amplification. This paired methodological study evaluated whether a pre-analytical chloroform:methanol cleanup step improves DNA extract quality, post-extraction amplifiability, and molecular detection of selected intestinal protozoa in cats. A total of 105 feline fecal samples were divided into paired aliquots and subjected either to direct automated DNA extraction or to extraction preceded by chloroform:methanol cleanup. DNA concentration and purity were assessed spectrophotometrically using DNA yield, A260/280, and A260/230 ratios. Extracts from both workflows were analyzed by PCR for Tritrichomonas foetus, Cryptosporidium spp., Pentatrichomonas hominis, and Giardia duodenalis. In addition, a post-extraction spike-recovery experiment was performed on selected paired extracts to directly evaluate PCR inhibition. Cleanup significantly increased DNA concentration and improved A260/280 and A260/230 ratios. It was also associated with additional Cryptosporidium and Giardia detections and significantly lower Cryptosporidium Ct values among paired positives. Spike-recovery testing showed that direct extracts markedly delayed amplification of an exogenous Cryptosporidium meleagridis 18S spike, whereas cleanup-based extracts showed significantly lower Ct delay and higher spike recovery. All Cryptosporidium-positive sequences were identified as Cryptosporidium felis. Sequencing of the six Giardia beta-giardin amplicons revealed four sequences assigned to assemblage F, one sequence assigned to assemblage B, and one sequence assigned to assemblage D. These findings support chloroform:methanol cleanup as a useful pre-analytical strategy for improving feline fecal DNA extract quality and reducing post-extraction PCR inhibition, although broader validation across additional extraction platforms and larger positive sample sets is required.