Evaluation of mtp40 genomic fragment amplification for specific detection of Mycobacterium tuberculosis in clinical specimens

Abstract

A PCR assay based on the species-specific mtp40 genomic fragment was developed for the specific detection and identification of Mycobacterium tuberculosis in different uncultured clinical specimens. The aim of the study was to evaluate the clinical applicability of this target DNA in comparison with those of conventional microbiological methods and to compare the results obtained with those obtained after amplification with the IS6110 repetitive element. Discrepant results were interpreted in conjunction with the patients' clinical data, medical histories, and responses to therapy. A total of 172 specimens from 162 patients with respiratory symptoms were tested, 101 specimens were obtained from 92 patients clinically suspected of having tuberculosis, and 71 specimens were obtained from 70 patients without known mycobacterial infection. The results of our study suggest that PCR amplification with the mtp40 genomic fragment provides a highly sensitive and specific technique for the detection of M. tuberculosis strains in clinical samples. It allows for the differentiation between M. tuberculosis and other related mycobacteria, including M. bovis, and is more specific than the IS6110 target. For these and other reasons, we propose that the mtp40 assay is a possible alternative for the specific direct detection of M. tuberculosis in clinical laboratories.

Evaluation of PCR in Detection of Mycobacterium tuberculosis from Formalin-Fixed, Paraffin-Embedded Tissues: Comparison of Four Amplification Assays

“…Again, the best sensitivities were shown by PCR3 and PCR4, which are based on the amplification of IS6110, a mobile genetic element usually present in multiple copies within the genomes of virtually all members of the M. tuberculosis complex (4,32,34,35). On the contrary, the amplification of the species-specific mtp40 region present in a single copy within the genome of M. tuberculosis (7,19), showed an overall low sensitivity when applied to formalinfixed tissues, which was opposite the result observed with fresh clinical specimens (11,15). The sensitivity of PCR can thus be possibly ameliorated by choosing as a target of the amplification DNA sequences likely to be present in multiple copies within the genome.…”

“…The primers used in the first round (outer primers) corresponded to nucleotides 9 to 25 (primer PT1; 5Ј-CAACGCGCCGTCGGTGG-3Ј) and 385 to 401 (primer PT2; 5Ј-CCCCCCACGGCACCGC-3Ј) of the mtp40 segment. These primers amplified a region of 396 bp (7,15). The primers used in the second round of PCR1 (inner primers) corresponded to nucleotides 44 to 65 (primer PT3; 5Ј-CACCACGTTAGGGATGCACTGC-3Ј) and 244 to 265 (primer PT4; 5Ј-CTGATGGTCTCCGACACGTTCG-3Ј) and amplified a region of 223 bp (11).…”

PCR for Detection of Mycobacterium tuberculosis in Experimentally Infected Dogs

“…The bacterial DNA from both strains was obtained from 30-days culture, growing on Lowenstein-Jensen medium without glycerol, following the method of Herrera and Segovia (1996). The DNA of M. tuberculosis strains was used as a positive reactions control.…”

“…Tissue samples were taken from lung, lymph nodes, liver, spleen, kidney and small intestine (5 g of each) of control and infected dogs. The samples preliminarily put in lysis buffer, with Stomacher 80 (Seward Limited, London, UK) were homogenized and DNA was extracted as described by Herrera and Segovia (1996). As positive control were used genome DNAs, isolated from strain 233/6195 and from a reference M. tuberculosis strain Aoyama NBMCC 7578.…”

Rapid diagnosis of tuberculosis directly from clinical specimens using a gene chip

“…hsp65, Rv0577, TbD1, Rv3120, Rv2073c, Rv1970, Rv3875, Rv0186, Rv0124, Rv3347c, Rv1510, mtp40, and mpb83) within the regions of difference of the bacterium. They were marked as M. tuberculosis-specific targets in the literature, in genomic databases, and in Medline [12][13][14][15][16][17][18][19][20]. We used PCR to construct the M. tuberculosis pattern as a reference.…”