Identification of Mycobacterium bovis Isolates by a multiplex PCR / Identificação de colônias isoladas de Mycobacterium bovis por PCR múltipla

Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates.

Colônias isoladas a partir de lesões sugestivas de tuberculose bovina foram testadas pela reação múltipla em cadeia da polimerase, usando oligonucleotídeos direcionados para as seqüências genômicas RvD1Rv2031c e IS6110, específicas para M. bovis e para o complexo Mycobacterium tuberculosis, respectivamente. A m-PCR identificou, com sucesso, 88,24% das colônias isoladas como M. bovis.

Evaluación de un ensayo de RPC múltiple para diferenciar micobacterias del complejo Mycobacterium tuberculosis en un laboratorio de referencia / Evaluation of a multiplex PCR assay to differentiate mycobacteria of the Mycobacterium tuberculosis complex in a reference laboratory

Mycobacteria that cause tuberculosis in animals and humans belong to the Mycobacterium tuberculosis complex. Techniques for conventional diagnosis are time-consuming and do not differentiate between different strains belonging to the M. tuberculosis complex. The aim of this study was to evaluate a multiplex PCR assay applicable to mycobacteria in culture with the capacity to differentiate different strains belonging to the M. tuberculosis complex in a reference laboratory. Primers based on genomics regions of difference (RD) consisting in DNA segments that are present in M. tuberculosis, but differentially deleted in several members of M. tuberculosis complex were used in a PCR assay. The test was applied to 86 clinical isolates of mycobacteria. The pattern of amplification allowed differentiating between M. tuberculosis, M. bovis and M. bovis BCG in a single PCR reaction. This PCR multiplex assay may be used in a Reference Laboratory of Tuberculosis Diagnosis as a complementary test to differentiate mycobacteria strains belonging to the M. tuberculosis complex. This test significantly reduces the time period between culture and strain identification, and thus for could favor the adoption of better strain specific antimycobacterial regimens as well as identification of zoonotic transmission of M. bovis to humans.

Las micobacterias que causan tuberculosis en animales y humanos pertenecen al complejo Mycobacterium tuberculosis. Las técnicas de diagnóstico convencional, además de ser lentas y laboriosas, no permiten diferenciar entre miembros de este complejo. El objetivo de este estudio fue evaluar ensayos de RPC múltiple para contribuir a la identificación diferencial de micobacterias del complejo M. tuberculosis a partir de cultivos, en un laboratorio de referencia. Se utilizaron oligonucleótidos partidores basados en regiones de diferencia (RD) que consisten en segmentos de ADN que están presentes en M. tuberculosis, pero que han sido eliminados diferencialmente del genoma de otros miembros del complejo M. tuberculosis. El ensayo se aplicó sobre 86 aislados clínicos de micobacterias. El patrón de amplificación permitió diferenciar entre cepas de M. tuberculosis, M. bovis y M. bovis variedad BCG en una única RPC. Este ensayo de RPC múltiple puede ser utilizado en el Laboratorio de Referencia de Diagnóstico de Tuberculosis como prueba complementaria para diferenciar micobacterias del complejo M. tuberculosis, contribuyendo a un acortamiento en el período de reporte de resultados y un tratamiento adecuado del paciente, y podría ser aplicado también en estudios epidemiológicos de transmisión zoonótica de M. bovis a humanos.

Oligonucleotide fingerprint analysis of a wild strain & its attenuated variant of Japanese encephalitis virus.

A strain of Japanese encephalitis (JE) virus was passaged serially through primary chick kidney cell cultures (45 times) and primary baby hamster kidney cell cultures (21 times). The resultant virus lost its lethal effect to 3 wk old mice by the ic route and 10 day old mice by the ip route. The oligonucleotide fingerprint analysis of the parent and the passaged strains showed 64 spots in common; 17 spots were present in the parent strain which were absent in the passaged virus, while the latter had acquired 9 spots which were not present in the parent virus.

Oligonucleotide fingerprint analysis of Japanese encephalitis virus strains of different geographical origin.

RNA fingerprint analysis was carried out with different strains of Japanese encephalitis virus which were isolated from Japan, China, India and Sri Lanka. From the similarity ratios, a similarity matrix was worked out which yielded a dendrogram. Geographical proximity of the place of isolation did not contribute much to the similarity of the fingerprint of the strains, nor did temporal proximity. The Japanese Nakayama strain had greater similarity with the Asansol strain from West Bengal. However, another strain from West Bengal, the Bankura strain, showed marked difference. Similarly the Bhopal and Beijing (China) strains were relatively close to each other while the Japanese JaGAr15460 strain was nearer to the strain from Gorakhpur. Serial mouse passage of the Asansol strain did not change the fingerprint pattern drastically.