title: Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine.
abstract: An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 10(7) to 10(8) Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.
authors.affiliation: Department of Bacteriology, Central Veterinary Institute, Lelystad, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Gerritsen
authors.forename: T
authors.initials: T
authors.lastname: Olyhoek
authors.forename: M A
authors.initials: MA
authors.lastname: Smits
authors.forename: B A
authors.initials: BA
authors.lastname: Bokhout
mesh_terms: D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis; D005260:Female; D007921:Leptospira interrogans / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D014556:Urine / Q000382:microbiology*; D014895:Weil Disease / Q000662:veterinary
chemical_list: D004269:DNA, Bacterial