title: High-throughput multiplex MIRU-VNTR typing of Mycobacterium bovis.
abstract: Spoligotyping is the most widely used method for genotyping Mycobacterium bovis (M. bovis). However, its discriminatory power varies widely between countries. MIRU-VNTR typing could be a promising alternative, although it generally requires the time consuming and laborious simplex PCR assays using standard agarose gel electrophoresis. The accuracy of this approach depends on good standardization and a certain degree of expertise. This study presents a version of MIRU-VNTR based on three triplex PCRs with automatic allelic assignation of the products analyzed in capillary electrophoresis. The technique was prospectively applied to 44 M. bovis and two Mycobacterium caprae (M. caprae) isolates, and 22 different MIRU-VNTRtypes were obtained; with spoligotyping, only 14 different types were obtained. The proposal makes it possible to shorten response times, automate procedures, and increase accuracy, thus minimizing errors in assigning genotypes. It would enable the switch from a standard limited method of genotyping M. bovis to a high-throughput discriminatory fingerprinting approach.
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain; CIBER Enfermedades Respiratorias, CIBERES, Spain.
authors.forename: Y
authors.initials: Y
authors.lastname: Navarro
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades Respiratorias, CIBERES, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Herranz
authors.affiliation: Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Romero
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CIBER Enfermedades Respiratorias, CIBERES, Spain; Departamento de Medicina, Facultad de Medicina, Universidad Complutense, Madrid, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Bouza
authors.affiliation: Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Domínguez
authors.affiliation: Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Madrid, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain; Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: de Juan
authors.affiliation: Servicio Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain; Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain; CEI Campus Moncloa, UCM-UPM, Madrid, Spain; CIBER Enfermedades Respiratorias, CIBERES, Spain. Electronic address: dgviedma2@gmail.com.
authors.forename: D
authors.initials: D
authors.lastname: García-de-Viedma
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D018598:Minisatellite Repeats / Q000235:genetics; D009163:Mycobacterium bovis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D013030:Spain; D014376:Tuberculosis / Q000235:genetics / Q000662:veterinary*
chemical_list: D004269:DNA, Bacterial
keywords: Genotyping; MIRU-VNTRtyping; Multiplex; Mycobacterium bovis