title: Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens.
abstract: A new quantitative real-time PCR (qPCR) assay based on Taqman® technology and minor groove binding (MGB) probe was developed for the diagnosis of leishmaniosis and quantification of Leishmania infantum DNA in infected dogs. This method was based on the amplification of a 122bp fragment of the highly conserved kDNA minicircles of L. infantum. The reaction was performed using the StepOnePlus™ system with StepOne software™. This assay was able to detect the presence of protozoan parasite DNA in amounts as low as 0.03 parasites per reaction. The standard curve designed for the quantification of parasites showed linearity over seven log DNA concentration range with a correlation coefficient >0.999 and both intra- and inter-assay variability demonstrated the high efficiency and reproducibility of the assay. The qPCR also proved to be successfully applicable to different clinical samples including blood, bone marrow, lymph node aspirates and conjunctival swabs.
authors.affiliation: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione di Bologna, 40127 Bologna, Italy. elena.galletti@izsler.it
authors.forename: Elena
authors.initials: E
authors.lastname: Galletti
authors.forename: Paolo
authors.initials: P
authors.lastname: Bonilauri
authors.forename: Lia
authors.initials: L
authors.lastname: Bardasi
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Fontana
authors.forename: Mattia
authors.initials: M
authors.lastname: Ramini
authors.forename: Maria
authors.initials: M
authors.lastname: Renzi
authors.forename: G
authors.initials: G
authors.lastname: Dosa
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Merialdi
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000469:parasitology; D003228:Conjunctiva / Q000469:parasitology; D018105:DNA, Kinetoplast / Q000032:analysis* / Q000097:blood / Q000235:genetics; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D018314:Leishmania infantum / Q000235:genetics / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D008198:Lymph Nodes / Q000469:parasitology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D018105:DNA, Kinetoplast