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Journal of Clinical Microbiology, January 1998, p. 239-242, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

Luz Elena Espinosa de los Monteros,1,dagger Juan Carlos Galán,1 Montserrat Gutiérrez,2 Sofía Samper,3 Juan F. García Marín,2 Carlos Martín,3 Lucas Domínguez,4 Luis de Rafael,1 Fernando Baquero,1 Enrique Gómez-Mampaso,1 and Jesús Blázquez1,*

Servicio de Microbiología, Hospital Ramón y Cajal, Instituto Nacional de la Salud,1 and Departamento de Patología Animal I (Sanidad Animal), Facultad de Veterinaria, Universidad Complutense,4 Madrid, Anatomía Patológica, Departamento de Patología Animal, Facultad de Veterinaria, Universidad de León, León,2 and Departamento de Microbiología y Medicina Preventiva, Facultad de Medicina, Universidad de Zaragoza, Zaragoza,3 Spain

Received 12 May 1997/Returned for modification 2 September 1997/Accepted 21 October 1997

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.

* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Ramón y Cajal, Ctra. Colmenar km 9.100, 28034 Madrid, Spain. Phone: 34-1-336 83 30. Fax: 34-1-336 88 09. E-mail: jesus.blazquez{at}hrc.es.

dagger Present address: Servicio de Microbiología, Hospital Infantil de México Federico Gómez, México 06720 D.F., México.

Journal of Clinical Microbiology, January 1998, p. 239-242, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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