title: Comparison of different commercial DNA extraction kits and PCR protocols for the detection of Echinococcus multilocularis eggs in faecal samples from foxes.
abstract: Effective and sensitive methods for the molecular detection of Echinococcus multilocularis in faecal samples of final hosts are crucial for the prevention and control of human alveolar echinococcosis and for studies on the epidemiology of the parasite. Little is known about the suitability of commercial test kits for isolating DNA of E. multilocularis from fox faeces and the performance of standard Polymerase Chain Reaction (PCR) protocols in relation to the quality of DNA extracted by these kits. We compared four different kits: ZR Faecal DNA MiniPrep™ (Zymo Research), FastDNA®SPIN Kit for Soil (MP Biomedicals), QIAamp®Fast DNA Stool Mini Kit (QIAGEN) and NucleoSpin®Soil Kit (Macherey-Nagel) for the extraction of DNA from E. multilocularis eggs present in faeces of foxes. Negative faecal samples were spiked with 600, 300, 150, 75, 37, 18, 9, 5 or 2 E. multilocularis eggs, and each egg concentration was tested 10 times with each of the DNA extraction kits. Each extracted DNA sample was amplified using three PCR protocols: i. conventional PCR (cPCR, Platinum®Taq, Invitrogen), ii. qPCR with the iQ™ Supermix (Bio-Rad) and iii. qPCR with the QuantiTect®Multiplex-Master Mix (QIAGEN). The highest analytical sensitivities for molecular detection of E. multilocularis eggs in spiked fox faeces were observed when combining either the QIAamp®Fast DNA Stool Mini Kit or the ZR Faecal DNA MiniPrep™ kit with the qPCR using the QuantiTect®Multiplex-Master Mix (Sensitivities 97% and 94%, respectively). Combinations including the remaining test kits (NucleoSpin®Soil Kit and FastDNA®SPIN Kit for Soil) showed a markedly lower analytical sensitivity for PCR examinations. The results of the present study indicate that it is of utmost importance to select suitable DNA extraction kits in combination with robust PCR methods or reagents to achieve acceptable analytical sensitivity in the molecular detection of E. multilocularis eggs in fox faecal samples.
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, NRL for Echinococcosis, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Pavlo.Maksimov@fli.de.
authors.forename: Pavlo
authors.initials: P
authors.lastname: Maksimov
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, NRL for Echinococcosis, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Gereon.Schares@fli.de.
authors.forename: Gereon
authors.initials: G
authors.lastname: Schares
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, NRL for Echinococcosis, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Sebastian.Press@fli.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Press
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, NRL for Echinococcosis, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Andreas.Frohlich@fli.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Fröhlich
authors.affiliation: Institute of Parasitology, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 266a, CH-8057, Zurich, Switzerland; Department of Farm Animals, Division of Swine Medicine, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland. Electronic address: Walter.Basso@access.uzh.ch.
authors.forename: Walter
authors.initials: W
authors.lastname: Basso
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, NRL for Echinococcosis, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Mandy.Herzig@fli.de.
authors.forename: Mandy
authors.initials: M
authors.lastname: Herzig
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, NRL for Echinococcosis, Südufer 10, 17493, Greifswald-Insel Riems, Germany. Electronic address: Franz.Conraths@fli.de.
authors.forename: Franz J
authors.initials: FJ
authors.lastname: Conraths
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification; D004443:Echinococcosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D048210:Echinococcus multilocularis / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology; D005260:Female; D005589:Foxes / Q000469:parasitology*; D010063:Ovum; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
chemical_list: D018509:DNA, Helminth; D011933:Reagent Kits, Diagnostic
keywords: Conventional PCR; Copro-inhibition; DNA isolation; Scat; Taeniid eggs; qPCR