title: A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.
abstract: Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples.
authors.affiliation: Laboratório de Micoplasmas, Departamento de Microbiologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo, Brazil; Laboratório de Microbiologia e Imunologia, Nucleo de Tecnologia em Saúde, Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, Brazil.
authors.forename: Lucas M
authors.initials: LM
authors.lastname: Marques
authors.forename: Aline T
authors.initials: AT
authors.lastname: Amorim
authors.forename: Hellen Braga
authors.initials: HB
authors.lastname: Martins
authors.forename: Izadora Souza
authors.initials: IS
authors.lastname: Rezende
authors.forename: Maysa Santos
authors.initials: MS
authors.lastname: Barbosa
authors.forename: Tassia Neves
authors.initials: TN
authors.lastname: Lobão
authors.forename: Guilherme B
authors.initials: GB
authors.lastname: Campos
authors.forename: Jorge
authors.initials: J
authors.lastname: Timenetsky
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis*; D005260:Female; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014509:Ureaplasma / Q000235:genetics*; D016869:Ureaplasma Infections / Q000175:diagnosis / Q000662:veterinary*; D014621:Vagina / Q000382:microbiology
chemical_list: D012336:RNA, Ribosomal, 16S
keywords: Bovines; Ureaplasma diversum; qPCR