title: A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine.
abstract: Leptospires are excreted in the urine of infected animals, and the prompt detection of leptospiral DNA using polymerase chain reaction (PCR) is increasingly being used. However, contradictory data has emerged concerning the diagnostic accuracy of the most popular PCR assays that target either the 16S ribosomal RNA (rrs) or the subsurface lipoprotein (LipL32) genes. In order to clarify the effect of the gene target, a novel hydrolysis probe-based, quantitative real-time PCR (qPCR) assay targeting the LipL32 gene was developed, validated, and then compared directly to the previously described rrs hydrolysis probe-based qPCR using a convenience collection of canine urine samples. The novel LipL32 qPCR assay was linear from 5.9 × 10(6) to 59 genome equivalents per reaction. Both the LipL32 and the rrs qPCR assays showed a limit of detection of 10 target copies per reaction indicating an approximately equivalent analytical sensitivity. Both assays amplified all 20 pathogenic leptospiral strains tested but did not amplify a representative collection of bacteria commonly found in voided canine urine. When the field samples were assayed, 1 and 5 out of 184 samples yielded an amplification signal in the LipL32 and rrs assays, respectively. Nevertheless, when the limit of detection was considered as the cutoff for interpreting findings, the 4 discordant cases were judged as negative. In conclusion, our study confirmed that both LipL32 and rrs are suitable targets for qPCR for the detection of leptospiral DNA in canine urine. However, the rrs target requires the mandatory use of a cutoff value in order to correctly interpret spurious amplifications.
authors.affiliation: Department of Veterinary Medical Sciences, Alma Mater Studiorum University of Bologna, Ozzano dell'Emilia, Bologna, Italy (Gentilini, Zanoni, Zambon)Genefast srl, Bazzano, Bologna, Italy (Turba) fabio.gentilini@unibo.it.
authors.forename: Fabio
authors.initials: F
authors.lastname: Gentilini
authors.affiliation: Department of Veterinary Medical Sciences, Alma Mater Studiorum University of Bologna, Ozzano dell'Emilia, Bologna, Italy (Gentilini, Zanoni, Zambon)Genefast srl, Bazzano, Bologna, Italy (Turba).
authors.forename: Renato Giulio
authors.initials: RG
authors.lastname: Zanoni
authors.affiliation: Department of Veterinary Medical Sciences, Alma Mater Studiorum University of Bologna, Ozzano dell'Emilia, Bologna, Italy (Gentilini, Zanoni, Zambon)Genefast srl, Bazzano, Bologna, Italy (Turba).
authors.forename: Elisa
authors.initials: E
authors.lastname: Zambon
authors.affiliation: Department of Veterinary Medical Sciences, Alma Mater Studiorum University of Bologna, Ozzano dell'Emilia, Bologna, Italy (Gentilini, Zanoni, Zambon)Genefast srl, Bazzano, Bologna, Italy (Turba).
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Turba
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000652:urine; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000652:urine; D004283:Dog Diseases / Q000175:diagnosis* / Q000382:microbiology / Q000652:urine; D004285:Dogs; D007919:Leptospira / Q000235:genetics / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis / Q000382:microbiology / Q000652:urine / Q000662:veterinary*; D008074:Lipoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000652:urine; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification / Q000652:urine; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; Leptospira; LipL32; cutoff value; dogs; quantitative real-time polymerase chain reaction