A rapid and accurate method for diagnosing mycobacterial infection is urgently needed in the clinical laboratory. This study describes the combined use of the BACTEC MGIT 960 system and the Invader assay. The Invader assay correctly identified and differentiated total 888 clinical and reference strains according to a target sequence. In particular, it detected and identified 116 (95.1%) of 122 positive liquid cultures soon after automatic positive signal of the MGIT 960 system. Thus, the Invader assay has the accuracy and sensitivity to be an effective procedure for a definitive diagnosis of mycobacterial infection in a clinical setting.
The key to any assay based on DNA sequences is the probe. The target region for DNA probes must be sufficiently conserved among clinical strains of the species, and the sequence data should be reliable, definitive, and commonly used. To identify mycobacteria, probes have traditionally been based on sequences from the 16S rRNA gene that corresponds to Escherichia coli
positions 130 to 210 and positions 430 to 500. However, the 16S rRNA genes of some species have the same or very similar sequences (5
). For example, using the first 500 bases of the 5′ end of the 16S rRNA gene to analyze sequences, RIDOM or MicroSeq (Applied Biosystems) (24
) cannot differentiate M. abscessus
and M. chelonae
; M. kansasii
and M. gastri
; M. marinum
and M. ulcerans
; M. senegalense
, M. houstonense
ATCC 49403, and M. farcinogenes
; M. porcinum
and M. neworleansense
ATCC 49404; and M. septicum
and M. peregrinum.
In the present study, probes were based on a combination of the 16S rRNA gene and the ITS-1 region. The specificity of these probes was confirmed by extensive comparison with well-studied databases.
Although the 16S rRNA gene has a low mutation rate, it does display microheterogeneity within a species. Microheterogeneity was examined by mixed probes within the variable regions of M. intracellulare (sequevars [sequence variants] I to V), and M. gordonae (sequevar I to V) (Table ). For example, M. intracellulare (sequevar III and IV) displayed microheterogeneity for type strain and were determined to be negative by the Amplicor M. intracellulare PCR. As an additional control for the accuracy of mycobacterial identification, mixed GNS probes were designed to select for the mycobacteria genus-specific region. These probes allowed differentiation of families closely related to the Mycobacteriaceae, including the Gordoniaceae, Nocardiaceae, and Tsukamurellaceae. A BRB probe based on the bacterial conserved region was designed for confirmation of the quantity and quality of sample DNA. In addition, mixed mycobacteria could be recognized by comparing the signal of a species probe with the signals of the BRB and GNS probes.
In the clinical laboratory, the MicroSeq 500 assay is a commercial sequencing assay which can be used for the routine identification of clinical mycobacterial isolates. The turnaround time for this assay is 2 days and requires approximately 4 h of a technologist's time. On the other hand, the turnaround time for an Invader assay of 20 samples was <6.5 h and required only about 0.5 h of a technologist's time. DNA sequencing requires less judgment on the part of technologists for interpretation and can identify a wide range mycobacterial species. However, labor, the reagents, equipment, and software necessary for this assay are significantly more expensive than the ribosomal probe hybridization. These factors limit the ability of hospital-based laboratories to use this assay (24
). Moreover, DNA sequencing requires more time and effort to target mixed cultures and multiple copy regions, such as ITS-1, since subcloning or separate cultures are needed. Setup of the Invader assay system requires no expensive measuring equipment, such as an automated DNA sequencer, and no exclusive workspace for PCR.
In the present study, the Invader probe setting cost-effectively detected more than 90% of the mycobacteria isolated in a routine clinical laboratory. On the other hand, each species probe could also perform an independent assay as needed.
In supplemental studies, although M. tuberculosis
complexes were difficult to identify at the species or strain level, these complex species and BCG strains could be discriminated by designing the Invader probes for gyrB
and RD1 (12
; data not shown). Since the Invader assay can measure genes with different GC contents, future versions may measure drug resistance genes simultaneously (41