Differential Identification of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria by Duplex PCR Assay Using the RNA Polymerase Gene (rpoB)

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,

_{Seong-Karp Hong,† Keun-Hwa Lee,}

_{Yeo-Jun Yun,}

_{Eui-Chong Kim,}

Young-Gil Park,

_{Gil-Han Bai,}

_{and Yoon-Hoh Kook}

_*

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 110-799,2_{and The Korean Institute of Tuberculosis, Korean}

National Tuberculosis Association, Seoul 137-140,3_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-bprpoBDNAs ofMycobacterium 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 differentiateM. tuberculosis

complex and NTM strains. In addition, restriction fragment length polymorphism analysis and direct sequenc-ing of the amplicon of NTM could be used to supplement species identification.

With the recent global resurgence of mycobacterial infec-tions, especially of tuberculosis, attributed to increased human immunodeficiency virus infection, there is an increasing de-mand for rapid, sensitive, and specific diagnostic methods for the detection and identification ofMycobacterium 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 ofM. tuber-culosis 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, usu-ally takes 4 to 6 weeks or longer (10). However, recent meth-odological advances in molecular biology have provided alter-native rapid approaches, e.g., the PCR and PCR-linked methods. For the rapid detection or identification ofM. tuber-culosis, 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 ofM.tuberculosisand/or NTM would be useful. For this pur-pose, 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 theM.

tuberculosiscomplex (6, 9) in the routine diagnostic laboratory by using Mycobacterium genus- and species-specific genes.

However, some of these genes have been found to lack spec-ificity forM. tuberculosis. In addition, IS6110PCR has been reported to produce false-negative (23) and false-positive (11) results, and themtp40gene is not present in allM.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.tuberculosiscomplex and NTM by using a single gene, the RNA polymerase␤-subunit-encoding gene (rpoB). To demon-strate 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 seventy-nine clinical isolates (193 M. tuberculosis complex [183 M.

tuberculosisand 10M.bovis] and 186 NTM isolates), which had been isolated by the Korean Institute of Tuberculosis and the Seoul National University Hospital, were identified by conven-tional 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 refer-ence 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 with-out 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 of the boiled mycobacterial suspension was used directly as a tem-plate for the DPCR assay.

* Corresponding author. Mailing address: Department of Microbi-ology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea. Phone: 82-2-740-8306. Fax: 82-2-743-0881. E-mail: [email protected].

† Present address: Department of Microbiology, College of Medi-cine, The Catholic University of Korea, Seoul 137-701, Korea.

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For construction of the two DPCR primer sets, rpoB se-quences of 44 mycobacteria (GenBank accession no. AF057449 to AF057493) were aligned by using the multiple alignment algorithm in the MegAlign package (Windows, ver-sion 3.12e; DNASTAR, Madison, Wis.). The specific nucleo-tides of theM.tuberculosisor 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 theM.tuberculosiscomplex 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) containing 2 U ofTaq poly-merase, 250 ␮M each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.3), and 1.5 mM MgCl2, and water was added to a final volume of 20␮l per reaction mixture. PCR was per-formed with an initial denaturation of 95°C for 5 min, 30 cycles of amplification (30 s at 95°C, 60 s at 72°C), and a final elon-gation at 72°C for 5 min (model 9600 thermocycler;

Perkin-Elmer Cetus). Denaturation was extended to 15 min for the clinical isolates.Escherichia coliDNA 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 di-rect sequencing for species identification. On the basis of the

rpoB sequences of mycobacteria (GenBank accession no. AF057449 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 mi-crocentrifuge tube, and water was added to a final volume of 20 ␮l per reaction mixture. Digestion was performed for 2 h at 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 se-quences, and by comparing these with sequences in the Gen-Bank database. Sequencing reactions with primers M5 and

TABLE 1. Mycobacteria and non-mycobacteria used for DPCR and their PCR amplification products

Species Type straind _clinicalNo. of

isolatesa

Size of amplicon

(bp) Species Type strain

d _clinicalNo. of

isolatesa

Size of amplicon

(bp)

Mycobacteria

M. abscessus CAP97E-03T ₈b ₁₃₆

M. africanum ATCC 25420T ₂₃₅

M. asiaticum ATCC 25276T ₁₃₆

M. aurum ATCC 23366T ₁₃₆

M. avium ATCC 25291T _{21 136}

M. bovis ATCC 19210T _{10 235}

M. bovisBCG French strain 1173P2T ₂₃₅

M. celatumType I ATCC 51131T ₁₃₆

M. celatumType II ATCC 51130T ₁₃₆

M. chelonae ATCC 35749T ₄b ₁₃₆

M. chitae ATCC 19627T ₁₃₆

M. fallax ATCC 35219T ₁₃₆

M. flavescens ATCC 14474T ₁₃₆

M. fortuitum ATCC 6841T _{18 136}

M. fortuitum49403 ATCC 49403 136

M. gastri ATCC 15754T ₁₃₆

M. genavense ATCC 51233T ₁₃₆

M. gordonae ATCC 14470T _{25 136}

M. haemophilum ATCC 29548T ₁₃₆

M. interjectum ATCC 51457T ₁₃₆

M. intermedium ATCC 51848T ₁₃₆

M. intracellulare ATCC 13950T _{47 136}

M. kansasii ATCC 12478T _{30 136}

M. leprae Thai53 strainT ₁₃₆

M. malmoense ATCC 29571T ₁₃₆

M. marinum ATCC 927T ₁₃₆

M. neoaurum ATCC 25795T ₁₃₆

M. nonchromogenicum ATCC 19530T ₁₃₆

M. paratuberculosis ATCC 19698T ₁₃₆

M. phlei ATCC 11758T ₁₃₆

M. peregrinum ATCC 14467T ₁₃₆

a_{Clinical isolates were identified by biochemical tests and analysis of partial 16S rDNA sequences.} b_{Differentiation ofM. abscessusandM. chelonaewas performed byrpoB(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.}

M. scrofulaceum ATCC 19981T _{15 136}

M. senegalense ATCC 35796T ₁₃₆

M. shimoidei ATCC 27962T ₁₃₆

M. simiae ATCC 25275T ₁₃₆

M. smegmatis ATCC 19420T ₁₃₆

M. szulgai ATCC 35799T _{18 136}

M. terrae ATCC 15755T ₁₃₆

M. thermoresistibile ATCC 19527T ₁₃₆

M. triviale ATCC 23292T ₁₃₆

M. tuberculosisH37Rv ATCC 27294T ₁₈₃c ₂₃₅

M. ulcerans ATCC 19423T ₁₃₆

M. vaccae ATCC 15483T ₁₃₆

M. xenopi ATCC 19250T ₁₃₆

Non-mycobacteria

Tsukamurella inchonensis KCTC 9866T ₁₃₆

Tsukamurella paurometabola KCTC 9821T ₁₃₆

Rhodococcus equi IMSNU 20114T ₁₃₆

Rhodococcus erythropolis IMSNU 20115 136 Rhodococcus rhodochrous IMSNU 20349T ₁₃₆

Nocardia brasiliensis KCTC 9136 136

Nocardia brevicatena KCTC 9370 136

Nocardia globerula KCTC 9001 136

Nocardia nova IMSNU 21197T ₁₃₆

Nocardia otitidiscaviarum IMSNU 21221T ₁₃₆

Corynebacterium diphtheriae MSNU NAe

Corynebacterium glutamicum IMSNU 21196T _NA

Staphylococcus aureus MSNU NA

Bacillus subtilis MSNU NA

Escherichia coli MSNU NA

Neisseria meningitidis MSNU NA

Haemophilus influenzae MSNU NA

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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 therpoBsequence analysis. Briefly, 16S rDNA fragments were amplified by using forward primer 285 and reverse primer 264 and then directly sequenced by using se-quencing 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 theM.tuberculosiscomplex or

NTM, respectively. When this method was applied to 17 non-mycobacteria, only 10 strains of Tuskamurella, Rhodococcus, andNocardiaspecies, which are phylogenetically close to my-cobacteria, produced a 136-bp DNA amplicon (Table 1). Fi-nally, 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 ofM.tuberculosis com-plex, the 136-bp DNAs were amplified from all of the 40 NTM strains (Fig. 2). Nothing was amplified from the negative con-trol,E.coliDNA. Therefore, DPCR assay allowed the differ-ential identification ofM.tuberculosiscomplex and NTM in a single reaction. Despite the mismatching of several nucleotides with those of the M5 or RM3 primer in therpoBsequences 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,rpoBDNAs were amplified from the serially diluted DNAs (10 ng to 10 fg) ofM.tuberculosisandM.avium.

FIG. 1. Locations (A) and sequences (B) of the DPCR primers on therpoBgene. DPCR products amplified by anM.tuberculosis-specific primer set (Tbc1-TbcR5) and an NTM-specific primer set (M5-RM3) were 235 and 136 bp long, respectively.rifr_{indicates the region associated} with the rifampin resistance ofM.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 ofE.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).

FIG. 2. DPCR assay results performed with the reference strains of

M.tuberculosiscomplex and NTM. Two amplicons of different sizes (235 and 136 bp) were amplified fromM.tuberculosiscomplex (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. nonchromogeni-cum; 11,M.triviale; 12,M.gordonae; 13,M.asiaticum; 14,M. thermore-sistibile; 15,E.coli(negative control).

TABLE 2. Differentiation ofM. tuberculosiscomplex and NTM clinical isolates by DPCR and conventional biochemical testing

DPCR product (size [bp])

No. of isolates identifieda_as:

M. tuberculosis

(n⫽193) (nNTM⫽186)

M. tuberculosisspecific (235) 193 0

NTM specific (136) 0 186

a_{Identification was separately performed by niacin and heat-stable catalase}

tests (pH 7.0, 68°C).

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The amplification product was obtained from 10 pg of M.

tuberculosisDNA, whereas in the case ofM. aviumDNA, 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 equiv-alents.

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 asM.tuberculosiscomplex and the other 186 strains were identified as NTM (Table 1). These results were com-pletely concordant with those obtained by conventional culture testing (Table 2). Although primer TbcR5 is located in therifr

region (20) (Fig. 1A), in which mutations are related to the rifampin resistance of M.tuberculosis, all of the 40 rifampin-resistant M. tuberculosis isolates produced 235-bp amplicons without PCR interference.

Since DPCR produces only one (136-bp)rpoBDNA from NTM, restriction analysis and sequencing could be used for further species identification. An algorithm for species identi-fication 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, exceptM.abscessus

andM.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 onrpoB sequences. Unlike previous methods, two dif-ferently sized DNAs were amplified from a single target gene of M. tuberculosisand NTM. The advantages of this DPCR assay over other multiplex PCRs are as follows. (i) rpoB nu-cleotides specific forM. tuberculosiscomplex or NTM are in-variably constant. Therefore, false-positive or -negative results

due to sequence variations do not occur. (ii) DPCR yields only one product, irrespective of theMycobacteriumspecies tested. Even the coexistence ofM.tuberculosisand NTM can be de-tected 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 onrpoB provides a rapid and reliable means for the differential identification of

M.tuberculosisand 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-PG6-01GM03-0002), and in part by the BK21 project for Medi-cine, Dentistry, and Pharmacy.

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