title: Allele-specific PCR method based on pncA and oxyR sequences for distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: intraspecific M. bovis pncA sequence polymorphism.
abstract: An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.
authors.affiliation: Servicio de Microbiología, Hospital Ramón y Cajal, Instituto Nacional de la Salud, Madrid, Spain.
authors.forename: L E
authors.initials: LE
authors.lastname: Espinosa de los Monteros
authors.forename: J C
authors.initials: JC
authors.lastname: Galán
authors.forename: M
authors.initials: M
authors.lastname: Gutiérrez
authors.forename: S
authors.initials: S
authors.lastname: Samper
authors.forename: J F
authors.initials: JF
authors.lastname: García Marín
authors.forename: C
authors.initials: C
authors.lastname: Martín
authors.forename: L
authors.initials: L
authors.lastname: Domínguez
authors.forename: L
authors.initials: L
authors.lastname: de Rafael
authors.forename: F
authors.initials: F
authors.lastname: Baquero
authors.forename: E
authors.initials: E
authors.lastname: Gómez-Mampaso
authors.forename: J
authors.initials: J
authors.lastname: Blázquez
mesh_terms: D000483:Alleles; D000581:Amidohydrolases / Q000235:genetics*; D004268:DNA-Binding Proteins; D009163:Mycobacterium bovis / Q000145:classification*; D009169:Mycobacterium tuberculosis / Q000145:classification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000581:Amidohydrolases; C022490:pyrazinamide deamidase