Determination of the sensitivity and specificity of PCR assays using different target dnas for the detection of Mycobacterium tuberculosis.

ABSTRACT

To establish a sensitive, specific and reproducible PCR assay for the detection of Mycobacterium tuberculosis, we evaluated three target DNAs IS6110, 65 kDa heat shock protein gene; and mtp40 genomic fragment. We purified genomic DNA from 15 mycobacterial strains including four M. tuberculosis isolates, four M. bovis BCG isolates, and one of each for M. fortuitum, M. avium, M. intracellulare, M. szulgai, M. scrofulaceum, M. chelonei, and M. gordonae from the culture and used them as the template DNA. We studied 3 primer sets for IS6110, 2 primer sets for 65 kDa, and 3 primer sets for mtp40. Depending on the assay, these primer sets were used in the single-step PCR and/or nested PCR. The PCR assay targeting the 65 kDa protein gene could detect all of the tested mycobacterial strains, whereas targeting the IS6110 sequence resulted in detection of only M. tuberculosis and M. bovis BCG. Furthermore, targeting the mtp40 genomic fragment could be used to distinguish M. tuberculosis from M. bovis BCG. Using a nested PCR assay with primer sets specifically targeting the IS6110 or 65 kDa, we have been able to detect single copy M. tuberculosis genomic DNA. When the mtp40 genomic fragment was used as the target DNA, the sensitivity of detection was 10 copies of M. tuberculosis genomic DNA. This assay was demonstrated to have good sensitivity and specificity for detection and discrimination of mycobacterial species, and could be used in analyzing the clinical samples with low copy number infections such as the cerebrospinal fluid from the patient with tuberculous meningitis.