Mycobacterium kansasii has been considerably pathogenic mycobacteria causing various human diseases with nontuberculous mycobacteria containing M. avium and M. intracellulare. It is the most common cause of nontuberculous mycobacterial lung disease in Europe [1, 2]. Detection and identification of mycobacteria is very important for epidemiological survey of disease and adequate drug use. Conventionally identification methods of mycobacteria are mainly biochemical tests of acid-fast isolates. These methods are not economical in view of time and expense. Lately, many molecular approaches for the identification of mycobacteria have been developed. Among them, The INNO-LiPA MYCOBACTERIA assay (LiPA; Innogenetics, Ghent, Belgium) using 16S？23S internal transcribed spacer (ITS) region is very specific and sensitive [3]. However, it is hard to identify some mycobacteria and has not yet been fully evaluated. Besides identification of mycobacteria using 16S rRNA gene, ITS, hsp65, sodA, and are sensitive and specific, but are too expensive [4].

In this study, novel M. kansasii-specific probe derived from rpoB was used to separately detect M. kansasii from 201 mycobacteria isolates.

Clinical isolates used in this study were provided by the Korean Institute of Tuberculosis and Department of Clinical Pathology, Seoul National University Hospital. Mycobacterial DNA samples were prepared by the bead beater-phenol extraction method [5].

A set of primers, which was previously used to amplify rpoB DNA (351 bp) encompassing the rif r (region associated with rifampin resistance in Mycobacteria) [5], was labeled with biotin and designated BioMF (5’biotin- CGACCACTTCGGCAACCG3’) and BioMR (5’biotin- TCGATCGGGCACATCCGG3’). Template DNA (approximately 50 ng) and 20 pmol of each primer (BioMF and BioMR) were added to a PCR mixture tube (Accu- Power PCR PreMix; Bioneer, Daejeon, Korea) containing 1 U of Taq DNA polymerase, 250 μM dNTP, 50 mM Tris？ HCl (pH 8.3), 40 mM KCl, 1.5 mM MgCl₂, and gel loading dye; the volume was then adjusted with distilled water to 20 μL. The reaction mixture was subjected to 30 cycles of amplification (5 minutes at 94℃, 1 minute at 95℃, 30 seconds at 68℃, 1 minute and 20 seconds at 72℃), and this was followed by a 10 minutes extension at 72℃.

DNA hybridization was performed as previously described [6], with minor modification (Fig. 1). Briefly, a oligonucleotide-specific probe, kanp (5’-GCC-AGC-TCTCCC- AGT-TCA-3’) was designed from the known rpoB sequences of M. kansasii [5].

Five picomoles of the probe DNAs was dissolved in 50 μL of immobilization buffer (1.5 M NaCl, 0.3 M Tris？HCl [pH 8.0], 0.3 M MgCl₂) and then dispensed into a microtiter well (NucleoLink Strips; Nunc, Rochester, NY, USA) and incubated over night at 37℃. The probe DNA mixture was then removed from the well and dried at 37℃ for 30 min. Wells were UV irradiated at 254 nm, for 5 minutes using an electronic UV crosslinker CEX-800 (Ultra-Lum, Claremont, CA, USA) and washed three times with 200 μL of washing buffer (1 M NaCl, 0.1 M Tris？HCl [pH 9.3], 2 mM MgCl₂, 0.1% Tween 20). Probe-coated wells was immediately stored at 4℃ and used for hybridization. One hundred microliters of hybridization solution (5 × SSC, 5 × Denhardt’s, 0.2% SDS, and 200 μg/mL of salmon sperm DNA) was dispensed into a probe-coated micro-titer well. Five microliters of the heat-denatured PCR product was mixed with the hybridization solution and incubated in the well for 30 minutes at 50℃. The mixture was then removed from the well, which was rinsed three times with 200 μL of 2 × SSC. One hundred microliters of alkaline phosphataseconjugated streptavidin solution (Amersham Life Science, Arlington Heights, IL, USA), diluted 1:2000 with incubation solution (0.3 M NaCl, 0.1 M Tris？HCl [pH 7.5] 2 mM MgCl₂, 0.05% Triton X-100), was then added to the well, and incubated for 15 minutes at room temperature. After incubation, the well was rinsed three times with 200 μL of incubation solution, then 100 μL of 1 M diethanolamine buffer (pH 9.8) containing 0.5 mM MgCl₂ and 10 mM p-nitrophenyl phosphate was added, and the whole solution was kept at room temperature for 60 min. The enzyme reaction was stopped using 5 μL of 10 M NaOH and the optical density at 405 nm (OD₄₀₅) of each well was read using a micro-titer plate reader (Multiskan Ascent; Labsystems, Grand Rapids, OH, USA). The OD₄₀₅ values of triplicated wells were use to draw a bar graph using SigmaPlot 2000 (Ver. 6.00) (SPSS Inc, Chicago, IL, USA).

The nucleotide sequences of the purified PCR products were directly determined as previously described [1].

For the sequencing reaction, 60 ng of PCR amplified DNAs, which were purified using a QIAEX II gel extraction kit (QIAGEN, Hilden, Germany), 5 pmol of either the forward or the reverse primer, and 4 μL of BigDye Terminator v2.0 100 RR mix (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) were mixed, and the contents were adjusted to a final volume of 10 μL with distilled water. The reaction was run for 30 cycles of 10 seconds at 96℃, 5 seconds at 60℃, and 4 minutes at 60℃. Both strands were sequenced as a crosscheck. Determined sequences were compared with those of reference strains in GenBank to compare sequence similarities.

This specific oligonucleotide probe, kanp probe, for M. kansasii were designed by basis on M. kansasii specific rpoB nucleotide sequence not found in other species of Mycobacterium. The OD₄₀₅ increased, depending on the concentration of coated this probe and its optimal concentration was evaluated at 5 pmol/well (Fig. 2). The average cut-off value of the colorimetric reaction was determined using the amplified rpoB DNAs of M. kansasii, which had been previously identified using culture-based methods.

This probe also was proved to be specific in terms of the amplification of the rpoB DNA of M. kansasii by PCRlinked DNA-DNA hybridization test (Fig. 3).

This probe was specific for subtype I, II, IV, and V of M. kansasii. There is no problem for detection and identification of M. kansasii from clinical samples because clinical pathogens are subtype I, and II of M. kansasii.

DNA-DNA hybridization test for detection and identification of M. kansasii, the average OD₄₀₅ for positive samples (n = 30) was 1.25 ± 0.32 and that of negative samples (n = 152) was 0.03 ± 0.02. Thus, the cut off value for hybridization test using kanp probe were set at 0.2. Reference strains other than members of the M. kansasii did not react with probes coated onto a micro-titer well plate. The DNA-DNA hybridization test was also performed with 201 clinical culture samples. Positive reactions, which mean M. kansasii were clearly identified. When kanp probe was used in DNA-DNA hybridization test, of the 201 strains, 27 strains (13.4%) produced positive results and 174 strains (86.6%) were negative (Table 1).

The sensitivity and specificity of this DNA hybridization method performed with culture samples was 100%. The negative strains by this DNA hybridization method were identified as M. tuberculosis (159 strains), M. avium (5 strains), M. intracellulare (8 strains), and M. flavescens (2 strain) by rpoB DNA sequence analysis.

There have been developed many diagnostic methods for mycobacterial infections containing conventional culture methods, PCR-based methods, and liquid culturebased mycobacterial detection systems, such the Bactec [7], MGIT [8], ESP [9], and BacT/Alert 3D [10]. There are many PCR-linked methods using 16S rRNA gene [11-14] and 16S？23S rRNA spacer region [3, 14] in mycobacteria. However, despite these efforts, a good standard protocol for detection and identification of M. kansasii has not yet been established because of various subtypes of M. kansasii.

In this study, we suggest that this DNA-DNA hybridization method assay using M. kansasii-specific rpoB DNA prob, kanp probe, could be useful for a rapid and precise detection for M. kansasii.