title: Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis.
abstract: The aim of the study was to develop a sensitive method using quantitative real-time polymerase chain reaction (qPCR) with pooled fecal samples for the screening of Johne's disease (JD). Manufacturer-specified and our new pooling method in combination with five commercial kits for DNA extraction and purification were compared. Different volumes of pooled fecal suspensions were tested, and the results were compared for individual samples and three pool sizes (5, 10, and 50 samples); each of the fecal suspensions, which were prepared from healthy dairy and beef cattle was spiked with 0, 10, 100, or 1000 cultured Mycobacterium avium subspecies paratuberculosis (MAP) organisms or was mixed with fecal suspensions from experimentally infected cattle. The MAP DNA detection proportion with our pooling method in combination with Johne-Spin kit (Fasmac, Japan) was 100% for all models and all pool sizes, except for the low shedder model with a pool size of 50. There was no loss of sensitivity in pools of 10 subjects or less by using the new method. These results suggest that new method is a sensitive, practical, and cost-effective screening test for the detection of MAP-infected cattle and the monitoring of JD-free herds.
authors.affiliation: Hygienics Division, National Livestock Breeding Center, Nishigo, Nishi-Shirakawa, Fukushima, 961-8511, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Mita
authors.affiliation: Division of Bacteriology and Parasitology, National Institute of Animal Health, Tsukuba, Ibaraki, 305-0856, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Hygienics Division, National Livestock Breeding Center, Nishigo, Nishi-Shirakawa, Fukushima, 961-8511, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: National Livestock Breeding Center, Okazaki Station, Okazaki, Aichi, 444-3161, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Tasaki
authors.affiliation: Hygienics Division, National Livestock Breeding Center, Nishigo, Nishi-Shirakawa, Fukushima, 961-8511, Japan.
authors.forename: Katsuo
authors.initials: K
authors.lastname: Utiyama
authors.affiliation: Hygienics Division, National Livestock Breeding Center, Nishigo, Nishi-Shirakawa, Fukushima, 961-8511, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Mori
mesh_terms: D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D005260:Female; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000302:isolation & purification; D010283:Paratuberculosis / Q000175:diagnosis* / Q000382:microbiology; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic
keywords: Commercial DNA extraction kits; IS900; Mycobacterium avium subspecies paratuberculosis; pooled fecal samples; real-time PCR; screening test