JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Nov. 2000, p. 4080–4085 Vol. 38, No. 11

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Detection and Identification of Mycobacteria by Amplification ofthe Internal Transcribed Spacer Regions with Genus-

and Species-Specific PCR PrimersHEEKYUNG PARK,1 HYUNJUNG JANG,1 CHEOLMIN KIM,2* BYUNGSEON CHUNG,2

CHULHUN L. CHANG,3 SOON KEW PARK,4 AND SUNDAE SONG5

Institute for Biomedical Research, SJ-Hightech Co., Ltd.,1 and Departments of Biochemistry,2 Clinical Pathology,3

and Internal Medicine,4 College of Medicine, Pusan National University, Pusan, and Institute ofClinical Research, National Masan Tuberculosis Hospital, Masan,5 Korea

Received 29 March 2000/Returned for modification 8 May 2000/Accepted 10 July 2000

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region foridentifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 speciesof mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and sevenpairs of mycobacterial species-specific primers were designed. All PCRs were performed under the sameconditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from theAmerican Type Culture Collection; 205 clinical isolates of mycobacteria, including 118 Mycobacterium tuber-culosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragmentsof approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosis complex-specific primersamplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with thepanmycobacterial primers and the M. tuberculosis complex-specific primers amplified two fragments that werespecific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-,M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primers amplified specificfragments only for the respective target organisms. These novel primers can be used to detect and identifymycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early andaccurate diagnosis of mycobacteriosis.

It is estimated that there are 8 million cases of tuberculosis(TB), causing 2.5 million deaths per year, worldwide, makingTB the foremost cause of death due to infection. Mycobacte-rial infections due to nontuberculous mycobacteria (NTM),such as the Mycobacterium avium complex (MAC), M. fortui-tum, and M. chelonae, are also on the increase (20). The in-creasing number of mycobacterial infections has made it clin-ically important to quickly identify mycobacteria at the specieslevel. The diagnosis of pathogenic versus nonpathogenic spe-cies not only has epidemiological implications but also is rel-evant for patient management (1).

PCR has proven to be a very useful tool for the rapid diag-nosis of infectious diseases, including mycobacteriosis. Many ofthe PCR assays used for detecting mycobacteria involve spe-cies-specific primers targeting the 16S rRNA, hsp65, 32-kDaprotein genes, or the internal transcribed spacer (ITS) anddetect only a single or a limited number of mycobacterialspecies (10, 13). The ITS between the l6S and 23S rRNA genesis approximately 270 to 360 bp but varies in size from speciesto species. It is considered to be a suitable target for probeswith which additional phylogenetic information can be derived(15). Furthermore, the ITS is suitable for differentiating spe-cies of mycobacteria and potentially can be used to distinguishclinically relevant subspecies (12). With respect to mycobacte-ria, both the high level of spacer sequence variation and thegood reproducibility of ITS sequencing suggest the applicabil-

ity of this approach. The purposes of this study were to designgenus-specific and species-specific primers and to determinethe PCR conditions for the simple and accurate detection ofclinically important mycobacterial species.

MATERIALS AND METHODS

Primer design. Conservative and polymorphic ITS sequences of mycobacteriawere sought in a multialignment of the ITS regions of 33 mycobacterial speciesusing CLUSTAL-W (http://genome.kribb.re.kr) (Table 1). The ITS sequences of31 mycobacterial species were obtained from GenBank. The ITS regions ofM. fortuitum and M. chelonae were cloned and sequenced, as there were nosequence data in GenBank, even though these species are frequently isolatedfrom clinical specimens (20). The sequence identity between the designed prim-ers and the mycobacterial ITS regions was analyzed with a BLAST search(http://www.ncbi.nlm.nih.gov). Based on the multialignment analysis data, a my-cobacterial genus-specific primer pair and seven species-specific primer pairswere designed (Table 2). A set of primers, ITS-F and mycom-2, was used toamplify partial ITS regions in mycobacteria. The two primers were designed fromthe highly conserved region on the basis of 16S rRNA and ITS sequences ofmycobacteria, respectively (3). Species-specific primers were designed from thepolymorphic regions of ITS sequences of mycobacteria.

Bacterial strains. The type strains of 20 mycobacterial species from the Amer-ican Type Culture Collection and 1l8 M. tuberculosis and 87 NTM clinical isolateswere used in this study. M. tuberculosis clinical isolates were randomly selectedfrom the stored strains at the mycobacterial laboratories of Pusan NationalUniversity Hospital and National Masan Tuberculosis Hospital. NTM (clinicalisolates) were identified by conventional methods and kindly provided by theKorean National Tuberculosis Association, which is the reference laboratory fortuberculosis diagnosis in eastern Asia. In the case of discrepancies betweentraditional and PCR methods, we confirmed our results by sequence analysis ofthe ITS. Fifty-five clinical isolates of nonmycobacterial pathogens were includedto confirm the specificity (Table 3). These isolates were identified biochemicallyor with commercial kits, such as API (Biomerieux, Marcy l’Etoile, France) andVitek (BioMerieux Vitek, Hazelwood, Mo.) kits.

Preparation of genomic DNA and PCR. All the mycobacteria were subculturedon Ogawa media, and nonmycobacteria were subcultured on blood agar plates.DNA was prepared from freshly grown colonies using an InstaGene matrix kit

* Corresponding author. Mailing address: Department of Biochem-istry, College of Medicine, Pusan National University, #10, Amidong-1-Ga, Seogu, Pusan 602-739, Korea. Phone: 82-51-240-7725. Fax: 82-51-248-1118. E-mail: kimcm@hyowon.cc.pusan.ac.kr.

4080

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

on A

pril 11, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

(Bio-Rad). PCR was performed with each pair of genus-specific primers andspecies-specific primers under the same conditions. Multiplex PCR was per-formed using panmycobacterial and M. tuberculosis complex-specific primers inthe same reaction tube. The primers were synthesized at a 50-nmol concentration(reagents were supplied by BioBasic Inc.). We tested the PCR conditions byadding various concentrations of tetramethylammonium chloride (TMAC), di-methyl sulfoxide, and glycerol to reduce nonspecific amplifications; finally, thefollowing conditions were chosen. The constituents of the PCR mixtures were asfollows: 500 mM KCl, 100 mM Tris HCl (pH 9.0), 1% Triton X-100, 0.2 mM eachdeoxynucleoside triphosphate (dATP, dGTP, dTTP, and dCTP), 1.5 mM MgCl2,10 mM TMAC, 10 pmol of each primer, and 1 U of Taq DNA polymerase(BioBasic). Each reaction was carried out for 5 min at 94°C; 30 cycles of 1 minat 94°C, 1 min at 60°C, and 1 min at 72°C; and 10 min at 72°C. The products wereelectrophoresed in 1.5% agarose gels. For multiplex PCR for the detection ofmycobacteria and M. tuberculosis, the primer concentration was optimized bymixing the ITS-F (10 pmol), mycom-2 (30 pmol), and TBF (20 pmol) primerstogether.

Nucleotide sequence accession numbers. The sequences determined in thisstudy were deposited in GenBank under accession numbers AF144326 andAF144327.

RESULTS

Designed primers. The ITS sequence has two conservedregions and several polymorphic regions in the 33 mycobacte-ria examined, including M. fortuitum and M. chelonae (Fig. 1).One pair of genus-specific and seven pairs of species-specificprimers for mycobacteria were designed after consideration ofproduct size and melting temperature (Table 3). The pair ofpanmycobacterial primers was expected to amplify specificfragments of 270 to 400 bp from species to species. Each pairof M. tuberculosis complex-, MAC-, M. fortuitum-, M. chelonae-,M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primerswas expected to amplify specific fragments of 121, 144, 223, 93,152, 99, and 105 bp in the respective target mycobacteria.

Detection of mycobacteria. For all the tested type strains ofmycobacteria, the specific fragments were amplified in the PCRwith the ITS-F and mycom-2 primers. The amplicons wereapproximately 270 to 400 bp in size, as expected (Fig. 2). For118 M. tuberculosis and 87 NTM clinical isolates, the expectedspecific fragments were amplified with genus-specific primers

TABLE 1. Strains used for sequence analysis andalignment of the ITS regions

Organism GenBank accession number(s)

M. avium LO7856, X74494, LO7848, LO7847,L15620, X74054, LO7858, LO7857,LO7855, LO7853, LO7852, LO7851,LO7850, LO7849, Z46421, and Z46422

M. conspicuum X92668M. farcinogenes Y10384M. gastri Y14182 and X97633M. genavense Y14183M. gordonae L42261, L42260, L42259, and L42258M. habana X74056M. intracellulare Z46425, X75602, X74057, L07859, and

Z46423M. kansasii X97632, L42262, and L42263M. leprae X56657M. lufu X74055M. malmoense Y14184 and Z35225M. marinum Y14185M. paratuberculosis X74495M. phlei X74493M. scrofulaceum L15622M. senegalense Y10385M. shimoidei AJ005005 and X99219M. simiae X75599, Y14186, Y14187, and Y14188M. smegmatis AB003598, AB003597, X76257, and U07955M. szulgai X99220M. terrae Z46427M. triplex Y14189M. triviale X99221M. tuberculosis complexa X58890, L15623, L26330, L26328, M20940,

and L26329M. ulcerans X99217M. xenopi Y14192, L15624, Y14190, and Y14191Mycobacterium species L15621M. fortuitumb AF144326M. chelonaeb AF144327

a Includes M. tuberculosis, M. africanum, M. bovis, and M. microti.b ITS sequences of these two species were not published in GenBank; they

were cloned and sequenced by us, and the sequences were submitted to Gen-Bank.

TABLE 2. Genus- and species-specific primers designed for the detection of mycobacteria

Primera Target species (expected size, bp) Position(s) Sequence

ITS-F Panmycobacteria (variable) 16S rRNA 59-TGGATCCGACGAAGTCGTAACAAGG-39mycom-2 ITS region 59-TGGATAGTGGTTGCGAGCAT-39

TBF M. tuberculosis (121) 28–47 59-TGGTGGGGCGTAGGCCGTGA-39TBR 129–148 59-CACTCGGACTTGTTCCAGGT-39

MACF MAC (144) 117–136 59-CCCTGAGACAACACTCGGTC-39MACR 241–260 59-GTTCATCGAAATGTGTAATT-39

FORF M. fortuitum (223) 45–64 59-CCGTGAGGAACCGGTTGCCT-39FORR 248–267 59-TAGCACGCAGAATCGTGTGG-39

CHEF M. chelonae (93) 59–78 59-GTTACTCGCTTGGTGAATAT-39CHER 133–152 59-TCAATAGAATTGAAACGCTG-39

GORF M. gordonae (152) 84–103 59-CGACAACAAGCTAAGCCAGA-39GORR 216–235 59-GCATCAAAATGTATGCGTTG-39

SCOF M. scrofulaceum (99) 131–150 59-TCGGCTCGTTCTGAGTGGTG-39SCOR 210–229 59-TAAACGGATGCGTGGCCGAA-39

SZUF M. szulgai (105) 124–143 59-AACACTCAGGCTTGGCCAGA-39SZUR 209–228 59-GAGGGCAGCGCATCCAATTG-39

a Primers are grouped in pairs.

VOL. 38, 2000 DETECTION AND IDENTIFICATION OF MYCOBACTERIA 4081

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

(ITS-F and mycom-2). Nonspecific amplicons were not seen inany of the nonmycobacterial pathogens tested (Table 3).

Identification of mycobacteria. PCR with the M. tuberculosiscomplex-specific primers, TBF and TBR, amplified an approx-imately 121-bp fragment only in the M. tuberculosis and M.bovis type strains (Fig. 3). As shown in Fig. 3, PCR with eachpair of NTM-specific primers amplified the specific fragmentof the expected size only in strains of the target organism. AllNTM clinical isolates were tested with each set of species-specific primers, and the specificity was confirmed.

Multiplex PCR. PCR with panmycobacterial and M. tuber-culosis complex-specific primers in the same tube amplified thetwo expected fragments, approximately 274 and 121 bp in size,in M. tuberculosis and M. bovis type strains and one panmyco-bacterial fragment in each of the NTM type strains (Fig. 4).The same result was also obtained for clinical isolates of M.tuberculosis and NTM. No fragment was found by PCR fornonmycobacterial pathogens.

DISCUSSION

Rapid identification of species of mycobacteria is an impor-tant factor for a successful diagnosis of mycobacteriosis. Itfacilitates selection of the appropriate drug therapy. However,it is not easy to identify species of mycobacteria, especiallyNTM. The Accuprobe culture confirmation kit (Gen-ProbeInc., San Diego, Calif.), one of the commercially availablemethods for mycobacterial identification, can be used for onlya limited number of mycobacterial species (13). Furthermore,the kit is relatively expensive. High-performance liquid chro-matography can also be used for mycobacterial identification,and its operating cost is low. However, the equipment is ex-pensive (7).

Currently, the widely accepted strategy formulated to im-prove methods for mycobacterial strain identification includesanalysis of the gene encoding 16S rRNA (15). Other targetgenes have been proposed for the identification of mycobac-teria by PCR-based sequencing. They included the spoligotyp-ing (spacer oligonucleotide typing) region (2, 21), the 32-kDaprotein gene (16), the dnaJ gene (18), the superoxide dis-mutase gene (22), the 65-kDa heat shock protein gene (hsp65)(14, 19), and the RNA polymerase gene (rpoB) (11). Eachtechnique has several advantages and disadvantages. For ex-ample, an excessive degree of variability, such as that found inthe hsp65 gene, may be undesirable, because such variability orinstability of species-specific signatures will make the develop-ment of reliable probes that cover all strains within a speciesimpossible (15). 16S rDNA sequences do not vary greatly withina species, and they are identical in some species (20). Moleculartyping by 16S rRNA sequence determination is not only morerapid but also more accurate than traditional typing (17).

Comparative sequence analysis of amplified rpoB DNAs canbe used efficiently to identify clinical isolates of mycobacteriain parallel with traditional culture methods and as a supple-ment to 16S rDNA gene analysis. For M. tuberculosis, rifampinresistance can be simultaneously determined (11).

This study demonstrated that an ITS-based PCR method hasa high degree of sensitivity and specificity for the detection andidentification of medically important species of mycobacteria.Glennon et al. (8) first speculated on its utility for the diagnosisof TB. The ITS sequence between the 16S rRNA and 23SrRNA genes, which is more variable than the 16S rRNA geneitself, has been shown to be species specific in many microor-ganisms (9). However, there is little between-species variationin the length of the spacer, which ranges from 235 nucleotidesfor M. xenopi to 285 nucleotides for M. gastri, a slowly growing

TABLE 3. Mycobacterial and nonmycobacterial isolates used in this study

Mycobacterial type strains andclinical isolates (no.) PCR resulta Nonmycobacterial clinical

isolates (no.) PCR resulta

M. abscessus ATCC 19977.......................................................... 1 Aeromonos hydrophila (2)........................................................... 2M. agri ATCC 27406 ................................................................... 1 Burkholderia cepacia (2) ............................................................. 2M. asiatticum ATCC 25276 ........................................................ 1 Candida albicans (2) ................................................................... 2M. austroafricanum ATCC 33464.............................................. 1 Citrobacter freundii (2) ................................................................ 2M. avium ATCC 25291............................................................... 1 Enterobacter aerogenes (2) .......................................................... 2M. bovis ATCC 19210................................................................. 1 Enterobacter cloacae (2).............................................................. 2M. chelonae ATCC 35752 .......................................................... 1 Enterococcus faecalis (2)............................................................. 2M. flavescens ATCC 14474......................................................... 1 Enterococcus faecium (2) ............................................................ 2M. fortuitum ATCC 6841............................................................ 1 Enterococcus raffinosis (2) .......................................................... 2M. gordonae ATCC 14470.......................................................... 1 Escherichia coli (2) ...................................................................... 2M. intracellulare ATCC 13950.................................................... 1 Klebsiella pneumoniae (2) ........................................................... 2M. kansasii ATCC 12478............................................................ 1 Plesiomonos shigelloides (2)........................................................ 2M. phlei ATCC 354 ..................................................................... 1 Proteus mirabilis (2)..................................................................... 2M. scrofulaceum ATCC 19981 ................................................... 1 Proteus vulgaris (2) ...................................................................... 2M. smegmatis ATCC 21701 ........................................................ 1 Providencia rettgeri (2)................................................................. 2M. szulgai ATCC 35799 .............................................................. 1 Pseudomonas aeruginosa (2)....................................................... 2M. terrae ATCC 15755................................................................ 1 Rahnella aquatilis (1) .................................................................. 2M. triviale ATCC 23292 .............................................................. 1 Salmonella spp. (2)...................................................................... 2M. tuberculosis H37Rv ................................................................ 1 Serratia marcescens (2)................................................................ 2M. vaccae ATCC 15483 .............................................................. 1 Shewanella putrefaciens (2)......................................................... 2MAC clinical isolates (31).......................................................... 1 Shigella flexneri (2)....................................................................... 2M. chelonae clinical isolates (10)............................................... 1 Shigella sonnei (2)........................................................................ 2M. fortuitum clinical isolates (13) .............................................. 1 Staphylococcus epidermidis (2) ................................................... 2M. gordonae clinical isolates (10) .............................................. 1 Staphylococcus aureus (2) ........................................................... 2M. scrofulaceum clinical isolates (3) ......................................... 1 Streptococcus agalactiae (2) ........................................................ 2M. szulgai clinical isolates (10) .................................................. 1 Streptococcus intermedius (2) ..................................................... 2M. terrae clinical isolates (10) .................................................... 1 Streptococcus pneumoniae (2) .................................................... 2M. tuberculosis clinical isolates (118) ........................................ 1 Vibrio paraphaelmolyticus (2) ..................................................... 2

a Result of PCR with ITS-F and mycom-2 primers: 1, PCR amplification; 2, no PCR amplification.

4082 PARK ET AL. J. CLIN. MICROBIOL.

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

FIG. 1. Alignment of the mycobacterial ITS sequences. The alignment includes conserved and polymorphic regions derived from the mycobacterial species. Theconserved sequences are in bold print, and the polymorphic sequences are in italic print. Dashes represent deletions, and asterisks represent identity.

FIG. 2. Genus-specific amplification of mycobacterial ITS by primers ITS-F and mycom-2. Lanes M, 100-bp size markers; lanes C, negative control; lane 1, M.abscessus ATCC 19977; lane 2, M. agri ATCC 27406; lane 3, M. asiaticum ATCC 25276; lane 4, M. austroafricanum ATCC 33464; lane 5, M. avium ATCC 25291; lane6, M. bovis ATCC 19210; lane 7, M. chelonae ATCC 35752; lane 8, M. flavescens ATCC 14474; lane 9, M. fortuitum ATCC 6841; lane 10, M. gordonae ATCC 14470;lane 11, M. intracellulare ATCC 13950; lane 12, M. kansasii ATCC 12478; lane 13, M. phlei ATCC 354; lane 14, M. scrofulaceum ATCC 19981; lane 15, M. smegmatisATCC 21701; lane 16, M. szulgai ATCC 35799; lane 17, M. terrae ATCC 15755; lane 18, M. triviale ATCC 23292; lane 19, M. tuberculosis H37Rv; lane 20, M. vaccaeATCC 15483.

VOL. 38, 2000 DETECTION AND IDENTIFICATION OF MYCOBACTERIA 4083

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

mycobacterium. The spacer sequences of slowly growing speciesare approximately 75 nucleotides shorter than those of rapidgrowers. Frothingham and Wilson (6) demonstrated intraspeciessequence polymorphisms in 4 of 11 species. M. gastri and M.avium each were split into two distinct sequevars (sequence vari-ations), designated Mga-A and Mga-B and Mav-A and Mav-B,

respectively, based on the nomenclature proposed by Frothing-ham and Wilson (6).

It was possible to develop genus- and species-specific prim-ers because the ITS has two conserved regions and severalpolymorphic regions in the 33 mycobacterial species. The twoconserved regions are close to each other within the mycobac-terial ITS (Fig. 1). The primers mycom-1 and mycom-2 weredesigned from these two conserved sequences. The ampliconsfor the primer set mycom-1 and ITS-R were expected to beapproximately 120 to 250 bp long. In a previous study, how-ever, our group demonstrated that these primers amplifiednonspecific bands in 28 clinical isolates of 14 species (3).Therefore, we used ITS-F and mycom-2 as a pair of genus-specific primers.

To develop multiplex PCR that will identify clinically impor-tant mycobacteria in one reaction tube, the PCR conditionsshould be the same in the individual reactions. It was mostdifficult to find conditions under which specific bands wereproduced successfully under the same PCR conditions, despitevariability in the length and GC content of each pair of prim-ers. We studied the influence of TMAC, dimethyl sulfoxide,and glycerol on the PCRs and found that the use of TMAC inthe PCR mixture dramatically reduced or eliminated nonspe-cific priming events, thereby enhancing the specificity of thereaction. In fact, TMAC binds selectively to dA-dT base pairs,altering the dissociation equilibrium and increasing the meltingtemperature (5). In a solution containing 3.0 M TMAC, thisdisplacement is sufficient to shift the melting temperature ofdA-dT base pairs to that of dG-dC base pairs. The PCR yieldincreased with 15 to 60 mM TMAC, while 150 mM TMACcompletely inhibited the reaction (4). In this study, PCR wascarried out in the presence of 0, 5, 10, 20, 50, and 100 mMTMAC. We observed an increase in PCR specificity at 10 mMTMAC (data not shown). A nonspecific upper band, of ap-proximately 550 bp, was seen with M. tuberculosis and M. bovis.We did not adjust the concentrations of target DNAs of typestrains and clinical isolates. Because we tested PCR with clin-ical isolates, target DNA was not constant. We optimized thePCR conditions.

In conclusion, the novel primers that we designed could beused to detect and identify mycobacteria simultaneously underthe same PCR conditions. Furthermore, this protocol facili-tated the early and accurate diagnosis of mycobacteriosis. Fur-ther experiments will be necessary to determine the conditionsneeded to detect mycobacteria directly from patient speci-mens, such as sputum. At the same time, the conditions forsuccessfully performing multiplex PCR using at least foursets of primers, including genus-specific and M. tuberculosis-,MAC-, and M. fortuitum-specific primers, should be studied.

ACKNOWLEDGMENT

This study was supported by a grant from the Korea Health R & DProject, Ministry of Health & Welfare, Republic of Korea (HMP-99-V-B-0004).

REFERENCES

1. American Thoracic Society. Diagnosis and treatment of disease caused bynontuberculous mycobacteria. This official statement of the American Tho-racic Society was approved by the Board of Directors, March 1997. MedicalSection of the American Lung Association. Am. J. Respir. Crit. Care Med.156:S1–25.

2. Aranaz, A., E. Liebana, A. Mateos, L. Dominguez, D. Vidal, M. Domingo, O.Gonzolez, E. F. Rodriguez-Ferri, A. E. Bunschoten, J. D. van Embden, andD. Cousins. 1996. Spacer oligonucleotide typing of Mycobacterium bovisstrains from cattle and other animals: a tool for studying epidemiology oftuberculosis. J. Clin. Microbiol. 34:2734–2740.

3. Chang, C. L., H. C. Son, S. H. Kim, C. M. Kim, B. S. Chung, S. K. Park,H. K. Park, H. J. Jang, and S. D. Song. 1999. Detection of mycobacteria by

FIG. 3. PCR with each pair of mycobacterial species-specific primers. Fromtop to bottom, primers TBF and TBR, MACF and MACR, FORF and FORR,CHEF and CHER, GORF and GORR, SZUF and SZUR, and SCOF andSCOR. Lanes M, 100-bp DNA ladder size markers; lane C, negative control; lane1, M. tuberculosis H37Rv; lane 2, M. bovis; lane 3, M. avium; lane 4, M. intracel-lulare; lane 5, M. fortuitum; lane 6, M. chelonae; lane 7, M. gordonae; lane 8, M.szulgai; lane 9, M. terrae; lane 10, M. scrofulaceum.

FIG. 4. Multiplex PCR with panmycobacterial and M. tuberculosis-specificprimers. Lanes M, 100-bp DNA ladder size markers; lane C, negative control;lane 1, M. tuberculosis H37Rv; lane 2, M. bovis; lane 3, M. avium; lane 4, M.intracellulare; lane 5, M. fortuitum; lane 6, M. chelonae; lane 7, M. gordonae; lane8, M. szulgai: lane 9, M. terrae; lane 10, M. scrofulaceum.

4084 PARK ET AL. J. CLIN. MICROBIOL.

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

amplifying the internal transcribed spacer regions with novel genus-specificprimers, p. 19–23. In Proceedings of XX World Congress of Pathology andLaboratory Medicine. Litosei-Rostignano-Bologna, Bologna, Italy.

4. Chevet, E., G. Lemaitre, and M. D. Katinka. 1995. Low concentrations oftetramethylammonium chloride increase yield and specificity of PCR. Nu-cleic Acids Res. 23:3343–3344.

5. Connors, T. D., T. C. Burn, T. VanRaay, G. G. Germino, K. W. Klinger, andG. M. Landes. 1997. Evaluation of DNA sequencing ambiguities using tetra-methylammonium chloride hybridization conditions. Biotechniques 22:1088–1090.

6. Frothingham, R., and K. H. Wilson. 1993. Sequence-based differentiation ofstrains in the Mycobacterium avium complex. J. Bacteriol. 175:2818–2825.

7. Glennon, M., M. G. Cormican, U. Ni Riain, M. Heginbothom, F. Gannon,and T. Smith. 1996. A Mycobacterium malmoense-specific DNA probe fromthe 16S/23S rRNA intergenic spacer region. Mol. Cell. Probes 10:337–345.

8. Glennon, M., T. Smith, M. Cormican, D. Noone, T. Barry, M. Maher, M.Dawson, J. J. Gilmartin, and F. Gannon. 1994. The ribosomal intergenicspacer region: a target for the PCR based diagnosis of tuberculosis. Tuber.Lung Dis. 75:353–360.

9. Gurtler, V., and V. A. Stanisich. 1996. New approaches to typing and iden-tification of bacteria using the 16S–23S rDNA spacer region. Microbiology142:3–16.

10. Kauppinen, J., R. Mantyjarvi, and M. L. Katila. 1999. Mycobacterium mal-moense-specific nested PCR based on a conserved sequence detected inrandom amplified polymorphic DNA fingerprints. J. Clin. Microbiol. 37:1454–1458.

11. Kim, B. J., S. H. Lee, M. A. Lyu, S. J. Kim, G. H. Bai, G. T. Chae, E. C. Kim,C. Y. Cha, and Y. H. Kook. 1999. Identification of mycobacterial species bycomparative sequence analysis of the RNA polymerase gene (rpoB). J. Clin.Microbiol. 37:1714–1720.

12. Lappayawichit, P., S. Rienthong, D. Rienthong, C. Chuchottaworn, A.Chaiprasert, W. Panbangred, H. Saringcarinkul, and P. Palittapongarnpim.1996. Differentiation of Mycobacterium species by restriction enzyme anal-ysis of amplified 16S–23S ribosomal DNA spacer sequences. Tuber. LungDis. 77:257–263.

13. Richter, E., S. Niemann, S. Rusch-Gerdes, and S. Hoffner. 1999. Identifica-tion of Mycobacterium kansasii by using a DNA probe (AccuProbe) andmolecular techniques. J. Clin. Microbiol. 37:964–970.

14. Ringuet, H., C. Akoua-Koffi, S. Honore, A. Varnerot, V. Vincent, P. Berche,J. L. Gaillard, and C. Pierre-Audigier. 1999. hsp65 sequencing for identifi-cation of rapidly growing mycobacteria. J. Clin. Microbiol. 37:852–857.

15. Roth, A., M. Fischer, M. E. Hamid, S. Michalke, W. Ludwig, and H. Mauch.1998. Differentiation of phylogenetically related slowly growing mycobacte-ria based on 16S–23S rRNA gene internal transcribed spacer sequences.J. Clin. Microbiol. 36:139–147.

16. Soini, H., and M. K. Viljanen. 1997. Diversity of the 32-kilodalton proteingene may form a basis for species determination of potentially pathogenicmycobacterial species. J. Clin. Microbiol. 35:769–773.

17. Springer, B., L. Stockman, K. Teschner, G. D. Roberts, and E. C. Bottger.1996. Two-laboratory collaborative study on identification of mycobacteria:molecular versus phenotypic methods. J. Clin. Microbiol. 34:296–303.

18. Takewaki, S., K. Okuzumi, I. Manabe, M. Tanimura, K. Miyamura, K.Nakahara, Y. Yazaki, A. Ohkubo, and R. Nagai. 1994. Nucleotide sequencecomparison of the mycobacterial dnaJ gene and PCR-restriction fragmentlength polymorphism analysis for identification of mycobacterial species. Int.J. Syst. Bacteriol. 44:159–166.

19. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bottger, and T. Bodmer.1993. Rapid identification of mycobacteria to the species level by polymerasechain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175–178.

20. Troesch, A., H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M.Kaplan, P. Cros, and C. Mabilat. 1999. Mycobacterium species identificationand rifampin resistance testing with high-density DNA probe arrays. J. Clin.Microbiol. 37:49–55.

21. Yo, G. S., H. L. Li, G. Torrea, A. Bunschoten, J. van Embden, and B. Gicquel.1997. Evaluation of spoligotyping in a study of the transmission of Mycobac-terium tuberculosis. J. Clin. Microbiol. 35:2210–2214.

22. Zolg, J. W., and S. Philippi-Schulz. 1994. The superoxide dismutase gene, atarget for detection and identification of mycobacteria by PCR. J. Clin.Microbiol. 32:2801–2812.

VOL. 38, 2000 DETECTION AND IDENTIFICATION OF MYCOBACTERIA 4085

on April 11, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

ERRATA

Detection and Identification of Mycobacteria by Amplification ofthe Internal Transcribed Spacer Regions with Genus-

and Species-Specific PCR PrimersHEEKYUNG PARK, HYUNJUNG JANG, CHEOLMIN KIM, BYNGSEON CHUNG,

CHULHUN L. CHANG, SOON KEW PARK, AND SUNDAE SONG

Institute for Biomedical Research, SJ-Hightech Co., Ltd., and Departments of Biochemistry, Clinical Pathology,and Internal Medicine, College of Medicine, Pusan National University, Pusan, and Institute of

Clinical Research, National Masan Tuberculosis Hospital, Masan, Korea

Volume 38, no. 11, p. 4080–4805, 2000. Page 4081, Table 2: The sequences for primers mycom-2, TBR, MACR, FORR, CHER,GORR, and SCOR should read 59-ATGCTCGCAACCACTATCCA-39, 59-ACCTGGAACAAGTCCGAGTG-39, 59-ATTACACATTTCGATGAACGC-39, 59-CCACACGATTCTGCGTGCTA-39, 59-TGCCAGCGTTTCAATTCTATTG-39, 59-CAACGCATACATTTTGATGC-39, and 59-TTCGGCCACGCATCCGTTTA-39, respectively.

PCR Detection of Granulocytic Ehrlichiae in Ixodes ricinus Ticksand Wild Small Mammals in Western Switzerland

JORGE S. LIZ, LAURENCE ANDERES, JOHN W. SUMNER, ROBERT F. MASSUNG, LISE GERN,BERNARD RUTTI, AND MICHEL BROSSARD

Department of Immunology and Department of Parasitology, Institute of Zoology, University of Neuchatel, 2007 Neuchatel,Switzerland; and Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National

Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Volume 38, no. 3, p. 1002–1007, 2000. Page 1002, abstract, line 6: “I. ricinus mammals” should read “I. ricinus ticks.”

828