A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences

BMC Veterinary Research

Methodology Article

Laura Betancor¹  Alessandra Piccirillo²  Alejandra Méndez³  Fernando Paolicchi³  Alejandra Velilla³  Claudia Morsella³  Ana Marandino⁴  Ruben Pérez⁴  Gonzalo Tomás⁴  Lucía Calleros⁴  Gregorio Iraola⁵ 

[1] Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay;Dipartimento di Biomedicina Comparata e Alimentazione, Università degli Studi di Padova, Padova, Italy;Laboratorio de Bacteriología, Unidad Integrada INTA-Universidad Nacional de Mar del Plata, Balcarce, Argentina;Sección Genética Evolutiva, Facultad de Ciencias, Iguá 4225, 11400, Montevideo, Uruguay;Sección Genética Evolutiva, Facultad de Ciencias, Iguá 4225, 11400, Montevideo, Uruguay;Unidad de Bioinformática, Institut Pasteur Montevideo, Montevideo, Uruguay;

关键词: Campylobacter fetus;    Molecular detection;    Real-time PCR;   

DOI  :  10.1186/s12917-016-0913-3

 received in 2016-02-20, accepted in 2016-12-06,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundCampylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains.ResultsWe developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability.ConclusionsThe possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species.

【 授权许可】

CC BY   
© The Author(s). 2016