Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR

Abstract

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.

FIG. 1.

Map of the amplified region from the 16S rDNA gene. Mycobacterium genus-conserved regions are shown in black. Hypervariable regions are striped. Primers and probes are also shown. Abbreviations: F, FAM; T, TAMRA; H, HEX; R, ROX.

FIG. 2.

Analysis of mycobacterial strain dilutions by fluorescence. Bars A, B, C, and D, 10⁴, 10³, 10², and 10 M. tuberculosis equivalents, respectively; E, F, G, H, and I, 10³ equivalents of M. kansasii, M. avium, M. intracellulare, M. xenopi, and M. fortuitum, respectively. The MTC probe was only positive in M. tuberculosis dilution samples.