Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

摘要: Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation feces and limited sensitivity since most kits use small amounts (<1 g). A direct PCR crude egg preparations was designed full compatibility with accurately quantify per gram (epg) determine composition. Eggs were recovered the flotation solution concentrated by sieving. Lysis achieved repeated boiling freezing cycles – Trichuris required additional mechanic disruption. Egg lysates directly used template Phusion polymerase which is particularly resistant inhibitors. Qualitative results obtained goats, cattle, horses, swine, cats, dogs mice. The finally established protocol also compatible quantitative real-time in presence EvaGreen no inhibition detectable when extracts diluted at least fourfold. Sensitivity comparable protocols spiked samples five epg reliably detected. For Toxocara cati a detection limit below one demonstrated. It possible distinguish T. canis using resolution melt (HRM) analysis, rapid tool identification. In human samples, restriction fragment length polymorphism (RFLP) HRM analysis discriminate Necator americanus Ancylostoma duodenale. method able significantly improve diagnosis increasing speed while decreasing overall costs. identification resistance alleles, products different post RFLP, reverse-line-blot, Sanger sequencing HRM.