Received 8 July 2003/Returned for modification 20 August 2003/Accepted 18 November 2003

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2004, p Vol. 42, No /04/$ DOI: /JCM Copyright 2004, American Society for Microbiology. All Rights Reserved. Differential Identification of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria by Duplex PCR Assay Using the RNA Polymerase Gene (rpob) Bum-Joon Kim, 1 Seong-Karp Hong, Keun-Hwa Lee, 1 Yeo-Jun Yun, 1 Eui-Chong Kim, 2 Young-Gil Park, 3 Gil-Han Bai, 3 and Yoon-Hoh Kook 1 * Department of Microbiology and Cancer Research Institute, Institute of Endemic Diseases, SNUMRC, Seoul National University College of Medicine, and Clinical Research Institute, 1 and Department of Clinical Pathology, Seoul National University College of Medicine, Seoul , 2 and The Korean Institute of Tuberculosis, Korean National Tuberculosis Association, Seoul , 3 Korea Received 8 July 2003/Returned for modification 20 August 2003/Accepted 18 November 2003 A novel duplex PCR method that can amplify the 235- and 136-bp rpob DNAs of Mycobacterium tuberculosis complex and nontuberculous mycobacteria (NTM), respectively, with two different sets of primers was used to differentially identify 44 reference strains and 379 clinical isolates of mycobacteria in a single-step assay. Showing 100% sensitivity and specificity, the duplex PCR method could clearly differentiate M. tuberculosis complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequencing of the amplicon of NTM could be used to supplement species identification. With the recent global resurgence of mycobacterial infections, especially of tuberculosis, attributed to increased human immunodeficiency virus infection, there is an increasing demand for rapid, sensitive, and specific diagnostic methods for the detection and identification of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) in a clinical setting (2, 3, 4). NTM infection can cause clinical problems, as its pathogenic potential and susceptibilities to antituberculosis treatments vary (22). In addition, mixed infections of M. tuberculosis and NTM have been reported (16). Therefore, it has become important to be able to differentiate between the two during the early stage of the diagnostic procedure. The diagnosis of mycobacterial infection is accomplished by culture-based identification. Primary culture of slowly growing mycobacteria, without using the BACTEC culture system, usually takes 4 to 6 weeks or longer (10). However, recent methodological advances in molecular biology have provided alternative rapid approaches, e.g., the PCR and PCR-linked methods. For the rapid detection or identification of M. tuberculosis, target genes specific to mycobacteria are used in a PCR (7, 8, 17, 19). Because the incidence of NTM infection is increasing, any methods capable of simultaneously determining the presence of M. tuberculosis and/or NTM would be useful. For this purpose, multiplex PCR, which simultaneously uses two or three different genes, has been frequently used, as the technique can specifically detect and identify different species of the genus Mycobacterium (6, 15, 18) and differentiate members of the M. tuberculosis complex (6, 9) in the routine diagnostic laboratory by using Mycobacterium genus- and species-specific genes. * Corresponding author. Mailing address: Department of Microbiology, Seoul National University College of Medicine, 28 Yongon- Dong, Chongno-Gu, Seoul , Korea. Phone: Fax: yhkook@plaza.snu.ac.kr. Present address: Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul , Korea. However, some of these genes have been found to lack specificity for M. tuberculosis. In addition, IS6110 PCR has been reported to produce false-negative (23) and false-positive (11) results, and the mtp40 gene is not present in all M. tuberculosis strains (21). These reports suggest that the multiplex PCR targeting of these genes has associated problems. In the present study, we used a simplified multiplex PCR assay, basically a duplex PCR (DPCR) assay, to differentiate M. tuberculosis complex and NTM by using a single gene, the RNA polymerase -subunit-encoding gene (rpob). To demonstrate the efficiency and usefulness of the DPCR assay in this context, we used it to identify reference strains and clinical isolates of mycobacteria. Forty-four mycobacteria and 17 non-mycobacteria were used as reference strains (Table 1). Three hundred seventynine clinical isolates (193 M. tuberculosis complex [183 M. tuberculosis and 10 M. bovis] and 186 NTM isolates), which had been isolated by the Korean Institute of Tuberculosis and the Seoul National University Hospital, were identified by conventional biochemical tests and provided for blind testing. An IS6110 PCR assay and analysis of partial 16S rrna gene (rdna) sequences were performed separately, and the results were compared with those of the DPCR assay. Two different kinds of DNA extraction protocols were used for type strains and clinical isolates. The DNAs of the reference strains were purified by using the bead beater-phenol extraction method as previously described (12, 13). The DNAs of clinical isolates were prepared by the boiling method without a purification step to reduce the DNA preparation time and to minimize the risk of cross contamination (1). A loopful culture of each sample was suspended in a screw-cap tube with 50 l of TEN buffer (10 mm Tris-HCl, 1 mm EDTA, 100 mm NaCl [ph 8.0]), and the tube contents were then boiled on a hot plate for 10 min. Without further purification, 5 l ofthe boiled mycobacterial suspension was used directly as a template for the DPCR assay. 1308

2 VOL. 42, 2004 NOTES 1309 TABLE 1. Mycobacteria and non-mycobacteria used for DPCR and their PCR amplification products Species Type strain d clinical No. of isolates a Size of amplicon (bp) Mycobacteria M. abscessus CAP97E-03 T 8 b 136 M. africanum ATCC T 235 M. asiaticum ATCC T 136 M. aurum ATCC T 136 M. avium ATCC T M. bovis ATCC T M. bovis BCG French strain 1173P2 T 235 M. celatum Type I ATCC T 136 M. celatum Type II ATCC T 136 M. chelonae ATCC T 4 b 136 M. chitae ATCC T 136 M. fallax ATCC T 136 M. flavescens ATCC T 136 M. fortuitum ATCC 6841 T M. fortuitum ATCC M. gastri ATCC T 136 M. genavense ATCC T 136 M. gordonae ATCC T M. haemophilum ATCC T 136 M. interjectum ATCC T 136 M. intermedium ATCC T 136 M. intracellulare ATCC T M. kansasii ATCC T M. leprae Thai53 strain T 136 M. malmoense ATCC T 136 M. marinum ATCC 927 T 136 M. neoaurum ATCC T 136 M. nonchromogenicum ATCC T 136 M. paratuberculosis ATCC T 136 M. phlei ATCC T 136 M. peregrinum ATCC T 136 Species Type strain d clinical No. of isolates a Size of amplicon (bp) M. scrofulaceum ATCC T M. senegalense ATCC T 136 M. shimoidei ATCC T 136 M. simiae ATCC T 136 M. smegmatis ATCC T 136 M. szulgai ATCC T M. terrae ATCC T 136 M. thermoresistibile ATCC T 136 M. triviale ATCC T 136 M. tuberculosis H37Rv ATCC T 183 c 235 M. ulcerans ATCC T 136 M. vaccae ATCC T 136 M. xenopi ATCC T 136 Non-mycobacteria Tsukamurella inchonensis KCTC 9866 T 136 Tsukamurella paurometabola KCTC 9821 T 136 Rhodococcus equi IMSNU T 136 Rhodococcus erythropolis IMSNU Rhodococcus rhodochrous IMSNU T 136 Nocardia brasiliensis KCTC Nocardia brevicatena KCTC Nocardia globerula KCTC Nocardia nova IMSNU T 136 Nocardia otitidiscaviarum IMSNU T 136 Corynebacterium diphtheriae MSNU NA e Corynebacterium glutamicum IMSNU T NA Staphylococcus aureus MSNU NA Bacillus subtilis MSNU NA Escherichia coli MSNU NA Neisseria meningitidis MSNU NA Haemophilus influenzae MSNU NA a Clinical isolates were identified by biochemical tests and analysis of partial 16S rdna sequences. b Differentiation of M. abscessus and M. chelonae was performed by rpob (306-bp) sequence analysis (13). c Composed of 40 rifampin-resistant and 143 rifampin-susceptible strains. d ATCC: American Type Culture Collection; KCTC: Korean Collection for Type Cultures; MSNU: Department of Microbiology, Seoul National University College of Medicine; IMSNU: Institute of Microbiology, Seoul National University. Superscript T indicates type reference strain. There were a total of 379 clinical isolates. e NA, not amplified. For construction of the two DPCR primer sets, rpob sequences of 44 mycobacteria (GenBank accession no. AF to AF057493) were aligned by using the multiple alignment algorithm in the MegAlign package (Windows, version 3.12e; DNASTAR, Madison, Wis.). The specific nucleotides of the M. tuberculosis or NTM strains were located at the 3 -hydroxyl end of each primer, as previously reported (12). The developed Tbc1 (5 -CGT ACG GTC GGC GAG CTG ATC CAA-3 )-TbcR5 (5 -C CAC CAG TCG GCG CTT GTG GGT CAA-3 ) and M5 (5 -G GAG CGG ATG ACC ACC CAG GAC GTC-3 )-RM3 (5 -CAG CGG GTT GTT CTG GTC CAT GAA C-3 ) primer sets amplify a 235-bp DNA sequence from the M. tuberculosis complex and a 136-bp DNA sequence from NTM, respectively (Fig. 1A). Primers (10 pmol of Tbc1-TbR5 and 20 pmol of M5-RM3) and 5 l of bacterial DNA were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer, Daejeon, Korea) containing2uoftaq polymerase, 250 M each deoxynucleoside triphosphate, 10 mm Tris-HCl (ph 8.3), and 1.5 mm MgCl 2, and water was added to a final volume of 20 l per reaction mixture. PCR was performed with an initial denaturation of 95 C for 5 min, 30 cycles of amplification (30 s at 95 C,60sat72 C), and a final elongation at 72 C for 5 min (model 9600 thermocycler; Perkin- Elmer Cetus). Denaturation was extended to 15 min for the clinical isolates. Escherichia coli DNA was used as a negative control. The PCR products were analyzed by agarose gel (1.5%) electrophoresis. After completion of the DPCR assay, all 186 NTM isolates showing a 136-bp DNA amplicon were further analyzed by restriction fragment length polymorphism (RFLP) and by direct sequencing for species identification. On the basis of the rpob sequences of mycobacteria (GenBank accession no. AF to AF057493), two restriction enzymes, MspI and HaeIII were selected by using MapDraw (version 3.14; DNASTAR). Two enzymes, MspI (TaKaRa, Shiga, Japan) and HaeIII (TaKaRa) were independently applied to the PCR products. Ten microliters of the PCR products, 2 U of each enzyme, and restriction buffer were transferred to a fresh microcentrifuge tube, and water was added to a final volume of 20 l per reaction mixture. Digestion was performed for 2hat 37 C. Following digestion, the mixtures were electrophoresed in a 3% agarose gel. NTM isolates were separately identified by determining 87-bp amplicon sequences, which excluded the primer sequences, and by comparing these with sequences in the Gen- Bank database. Sequencing reactions with primers M5 and

3 1310 NOTES J. CLIN. MICROBIOL. FIG. 1. Locations (A) and sequences (B) of the DPCR primers on the rpob gene. DPCR products amplified by an M. tuberculosis-specific primer set (Tbc1-TbcR5) and an NTM-specific primer set (M5-RM3) were 235 and 136 bp long, respectively. rif r indicates the region associated with the rifampin resistance of M. tuberculosis. Nucleotides of four mycobacteria differing from those of the primers are shown in bold letters. Numbers indicate the amino acid position of the RNA polymerase subunit of E. coli. Note that TbcR5 and RM3 are displayed in reverse direction. TB, M. tuberculosis H37Rv (GenBank accession no. AF057454); AVI, M. avium (GenBank accession no. AF057457); INT, M. intracellulare (GenBank accession no. AF057472); KAN, M. kansasii (GenBank accession no. AF057473). RM3 were performed as previously described (13). Analysis of the partial 16S rdna sequence was performed separately to identify the NTM strains, and the results were compared with the results of the rpob sequence analysis. Briefly, 16S rdna fragments were amplified by using forward primer 285 and reverse primer 264 and then directly sequenced by using sequencing primer 244, as previously described (14). The specificity of each primer set was assessed by a separate PCR. When a PCR using each specific primer set (Tbc1-TbcR5 or M5-RM3) was applied to the 44 reference strains at the same annealing temperature, an amplicon of either 235 or 136 bp of DNA was observed from the M. tuberculosis complex or NTM, respectively. When this method was applied to 17 nonmycobacteria, only 10 strains of Tuskamurella, Rhodococcus, and Nocardia species, which are phylogenetically close to mycobacteria, produced a 136-bp DNA amplicon (Table 1). Finally, a DPCR assay with a mixture of the two primer sets was performed on the reference strains. While the 235-bp DNAs were amplified from only four strains of M. tuberculosis complex, the 136-bp DNAs were amplified from all of the 40 NTM strains (Fig. 2). Nothing was amplified from the negative control, E. coli DNA. Therefore, DPCR assay allowed the differential identification of M. tuberculosis complex and NTM in a single reaction. Despite the mismatching of several nucleotides with those of the M5 or RM3 primer in the rpob sequences of NTM (Fig. 1B), all of the NTM strains tested produced 136-bp amplicons. In order to determine the lower limit of the sensitivity of the DPCR method, rpob DNAs were amplified from the serially diluted DNAs (10 ng to 10 fg) of M. tuberculosis and M. avium. FIG. 2. DPCR assay results performed with the reference strains of M. tuberculosis complex and NTM. Two amplicons of different sizes (235 and 136 bp) were amplified from M. tuberculosis complex (lanes 1 to 4) and NTM (lanes 5 to 14) strains by a single DPCR. Lanes: M, marker DNA (50-bp ladder); 1, M. tuberculosis; 2, M. africanum; 3, M. bovis; 4, M. bovis BCG; 5, M. avium; 6, M. paratuberculosis; 7, M. scrofulaceum; 8, M. intracellulare; 9, M. terrae; 10, M. nonchromogenicum; 11, M. triviale; 12, M. gordonae; 13, M. asiaticum; 14, M. thermoresistibile; 15, E. coli (negative control). TABLE 2. Differentiation of M. tuberculosis complex and NTM clinical isolates by DPCR and conventional biochemical testing DPCR product (size [bp]) No. of isolates identified a as: M. tuberculosis (n 193) NTM (n 186) M. tuberculosis specific (235) NTM specific (136) a Identification was separately performed by niacin and heat-stable catalase tests (ph 7.0, 68 C).

4 VOL. 42, 2004 NOTES 1311 FIG. 3. Algorithm for species identification by DPCR-RFLP (136-bp amplicons) with MspI and HaeIII. The amplification product was obtained from 10 pg of M. tuberculosis DNA, whereas in the case of M. avium DNA, as little as 1 pg was detected on an ethidium bromide-stained gel (data not shown). Taking 5 fg of DNA as a mycobacterial cell equivalent (5), this method could detect 2,000 bacillus equivalents. The usefulness of the DPCR assay was demonstrated by applying the technique to the identification of 379 clinical isolates. Of the 379 culture isolates examined, 193 strains were identified as M. tuberculosis complex and the other 186 strains were identified as NTM (Table 1). These results were completely concordant with those obtained by conventional culture testing (Table 2). Although primer TbcR5 is located in the rif r region (20) (Fig. 1A), in which mutations are related to the rifampin resistance of M. tuberculosis, all of the 40 rifampinresistant M. tuberculosis isolates produced 235-bp amplicons without PCR interference. Since DPCR produces only one (136-bp) rpob DNA from NTM, restriction analysis and sequencing could be used for further species identification. An algorithm for species identification by DPCR-linked restriction analysis with MspI and HaeIII was developed (Fig. 3). All of the 186 NTM isolates analyzed in this study were tentatively identified in accordance with this scheme. However, NTM isolates, except M. abscessus and M. chelonae, which have identical sequences in the 87-bp rpob region, could be exactly identified by comparing their determined rpob sequences to those of reference strains. These results are concordant with those obtained by 16S rdna analysis and culture-based biochemical testing. In the present study, we developed a novel DPCR assay based on rpob sequences. Unlike previous methods, two differently sized DNAs were amplified from a single target gene of M. tuberculosis and NTM. The advantages of this DPCR assay over other multiplex PCRs are as follows. (i) rpob nucleotides specific for M. tuberculosis complex or NTM are invariably constant. Therefore, false-positive or -negative results due to sequence variations do not occur. (ii) DPCR yields only one product, irrespective of the Mycobacterium species tested. Even the coexistence of M. tuberculosis and NTM can be detected by the presence of two different PCR products in a single reaction mixture. (iii) Moreover, the (136-bp) rpob DNAs of NTM can be further analyzed by RFLP or by direct sequencing to supplement species identification. In conclusion, the DPCR assay based on rpob provides a rapid and reliable means for the differential identification of M. tuberculosis and NTM in culture with a single reaction. B.-J. Kim and S.-K. Hong contributed equally to this work. This study was supported by a grant from the Korean Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01- PJ10-PG6-01GM ), and in part by the BK21 project for Medicine, Dentistry, and Pharmacy. REFERENCES 1. Afghani, B., and H. R. Stutman Polymerase chain reaction for diagnosis of M. tuberculosis: comparison of simple boiling and a conventional method for DNA extraction. Biochem. Mol. Med. 57: Barnes, P. F., A. B. Bloch, P. T. Davison, and D. E. Sneider, Jr Tuberculosis in patients with human immunodeficiency virus infection. N. Engl. J. Med. 324: Bloom, B. R Back to a frightening future. Nature (London) 358: Bloom, B. R., and C. J. L. Murray Tuberculosis: commentary on a reemergent killer. Science 257: Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry III, F. Tekaia, K. Badcock, D. Basham, D. Brown, et al Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: Del Portillo, P., M. C. Thomas, E. Martínez, C. Marañon, B. Valladares, M. E. Patarroyo, and M. C. López Multiprimer PCR system for differential identification of mycobacteria in clinical samples. J. Clin. Microbiol. 34: Eing, B. R., A. Becker, A. Sohns, and R. Ringelmann Comparison of RocheCobas Amplicor Mycobacterium tuberculosis assay with in-house PCR and culture for detection of M. tuberculosis. J. Clin. Microbiol. 36: Gunisha, P., H. N. Madhavan, U. Jayanthi, and K. L. Therese Polymerase chain reaction using IS6110 primer to detect Mycobacterium tuberculosis in clinical samples. Indian J. Pathol. Microbiol. 44: Herrera, E. A., O. Perez, and M. Segovia Differentiation between

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