Mycobacterium Tuberculosis
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JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1990, p. 2200-2204 Vol. 28, No. 10 0095-1137/90/102200-05$02.00/0 Copyright © 1990, American Society for Microbiology Polymerase Chain Reaction for Detection of Mycobacterium tuberculosis ULF SJOBRING,l MICHAEL MECKLENBURG,"2 ASE BENGÂRD ANDERSEN,3 AND HÂKAN MIORNERl* Department of Medical Microbiologyl and Department ofPure and Applied Biochemistry,2 University ofLund, Lund, Sweden, and Mycobacteria Department, Statens Seruminstitut, Copenhagen, Denmark3 Received 6 March 1990/Accepted 25 June 1990 A polymerase chain reaction for the specific detection of mycobacteria belonging to the Mycobacterium tuberculosis complex was developed. Using a single primer pair derived from the nucleotide sequence of protein antigen b of M. tuberculosis, we achieved specific amplification of a 419-base-pair DNA fragment in M. tuberculosis and M. bovis. After DNA was extracted from mycobacteria by using a simple, safe lysis procedure, we detected the 419-base-pair sequence in samples containing few mycobacteria. Preliminary data suggested that this technique could be applied to clinical specimens for early and specific diagnosis of tuberculosis. Several mycobacterial species are associated with dis- to detect DNAs extracted from various mycobacteria (3, 7). eases in humans. Tuberculosis is caused by Mycobacterium The latter application is based on the sequences of the heat tuberculosis, Mycobacterium africanum, or Mycobacterium shock proteins of M. bovis bacillus Calmette-Guérin (BCG) bovis and is a major health problem, particularly in devel- (21) and M. tuberculosis (18). These sequences are shared by oping countries. The number of cases of tuberculosis world- many other species, and labeled probes are required to wide is estimated to be some 30 million, with an annual identify amplified DNA specific for M. tuberculosis. incidence of 10 million (9). Mycobacteria other than M. The nucleotide sequence of the 38-kilodalton protein an- tuberculosis are widely distributed in nature, and infections tigen b (Pab) of M. tuberculosis was recently published (2). with mycobacteria other than M. tuberculosis are a medi- When serological methods were used, this protein could be cally important problem (4, 22). The emergence of acquired detected only in M. tuberculosis and M. bovis. Thus, hyper- immunodeficiency syndrome has also resulted in an in- immune sera from rabbits immunized with other mycobac- creased incidence of mycobacterial infections (20). terial species did not bind affinity-purified Pab (10, 23). This Bacteriological diagnosis of mycobacterial infections re- species restriction makes Pab a suitable candidate for diag- mains a problem, and there is an urgent need for a rapid and nostic purposes. In this paper we describe a PCR which was reliable diagnostic test. Culturing of organisms has a speci- used for detection of M. tuberculosis and which was based ficity that approaches 100% and permits susceptibility test- on the nucleotide sequence of the Pab gene. ing of the isolates, but the slow growth of most pathogenic mycobacteria (3 to 6 weeks) results in delays in diagnosis. MATERIALS AND METHODS When few mycobacteria are present at the site of infection, Bacterial strains and growth conditions. The bacterial multiple cultures may also be necessary to ensure a positive strains which we used are listed in Table 1. The mycobac- result. Identification of acid-fast organisms in stained smears terial strains were kindly provided by Veronique Lévy- may provide rapid diagnosis, but this method does not allow Frébault, Institut Pasteur, Paris, France, and by J. Stanford. identification at the species level and requires that a rela- M. bovis BCG was obtained from the Institut Pasteur, Paris, tively large number of bacteria (>104/ml) be present in the France. Mycobacteria were grown in Middlebrook 7H9 sample (6). Serological techniques may be useful in some broth (Difco Laboratories, Detroit, Mich.) supplemented clinical situations, but both the sensitivity and the specificity with OADC enrichment or on Middlebrook 7H10 agar (Dif- of these tests are unsatisfying (5). Gas chromatography with co) for 1 to 3 weeks at 37°C. During the first week the flame ionization detection has been shown to be useful for cultures were incubated in an atmosphere containing 5% the identification of cultured mycobacteria, but this method C02, and thereafter the cultures were incubated in air. To is not sensitive enough to be used for clinical specimens (12). obtain samples containing a known number of viable cells, Mass spectrometric detection has been used successfully to M. bovis BCG was cultured in Middlebrook 7H9 medium detect tuberculostearic acid in clinical samples but is an containing 0.05% Tween 80 and harvested at the exponential elaborate technique which requires expensive equipment growth phase. The suspended bacteria were mixed with a (13). The DNA or RNA hybridization tests with labeled vortex mixer for 10 min and then were allowed to sediment specific probes which have been described so far are not for 1 h. The supernatant was decanted and briefly sonicated sensitive enough to be used for clinical specimens without in a water bath, and then serial 10-fold dilutions were made prior culturing (15). in phosphate-buffered saline containing 0.05% Tween 80. The advent of the polymerase chain reaction (PCR) (16, Samples (100 pl) from the dilutions were inoculated onto 17) has opened new possibilities for diagnosis of microbial Middlebrook 7H10 agar, and the preparations were incu- infections. This technique has already been applied to a bated for 3 weeks at 37°C to determine the number of viable number of microorganisms. In the field of mycobacteria the organisms present. PCR has been used to identify Mycobacterium leprae (8) and Isolation of DNA. Mycobacterial DNA was prepared es- sentially as described by Baess (1). Bacteria grown in liquid cultures were lysed with sodium dodecyl sulfate, and the * Corresponding author. proteins were removed with proteinase K. Cell debris, 2200 VOL. 28, 1990 PCR FOR DETECTION OF M. TUBERCULOSIS 2201 TABLE 1. Specificity of the PCR based on the DNA sequence in 50 mM Tris hydrochloride (pH 8.0), and boiled for 5 min. of Pab of M. tuberculosis A 100-pul portion of each cell suspension was mixed with 25 pul of acid-washed glass beads (diameter, <150 ,um; Sigma SpeciesSpeciesor prepn (type culture) Presence~~~~~~~PCRofproductthe 419-bp Chemical Co., St. Louis, Mo.) suspended in 50 mM Tris hydrochloride. The mixture was then subjected to ultrason- Mycobacterium tuberculosis (H37Rv) ............ + M. bovis (ATCC 19210) .............................. + ication at 20 kHz in a water bath (Schoeller & Co., Frank- M. bovis BCG ................................... + furt, Federal Republic of Germany) for 15 min at 50°C; 10 pul M. kansasii (HB4962). of the supernatant was used as the target DNA in the M. marinum (ATCC 927) . amplification assay. M. scrofulaceum (HB1565). Clinical samples. Seven clinical samples were selected at M. szulgai (NCTC 10831). random from among smear-positive and smear-negative M. gordonae (ATCC 14470). samples. The sputum samples were treated with dithiothre- M. flavescens (NCTC 10271) . M. xenopi (HB1895). itol (Sputolysin; Behring Diagnostics, La Jolla, Calif.), M. avium (NCTC 8551) . whereas the samples from bronchial and gastric lavages were M. intracellulare (ATCC 13950) . not treated. The specimens were centrifuged at 2,300 x g for M. gastri (W471) . 20 min, the supernatants were discarded, and the pellets M. malmoense (ATCC 29571). were suspended in approximately 250 pul of 50 mM Tris M. nonchromogenicum (NCTC 10424). hydrochloride (pH 8.0). A 100-,ul portion of each of the M. terrae (W45) . samples was boiled. DNA was extracted as described above. M. triviale (T-254-3) . Selection of primers. The sequences of oligonucleotides M. paratuberculosis (ATCC 43015). MT1 and MT2, which were used as primers in the PCR, and M. fortuitum (HB1792) . the sequence of oligonucleotide MT3, which was used as a M. chelonae subsp. abscessus . M. phlei (NCTC 10266). probe to identify the amplified DNA in DNA hybridization M. smegmatis (ATCC 14468). experiments, were homologous to sequences in a 419-base- M. vaccae (ATCC 15483). pair (bp) region of the gene encoding Pab of M. tuberculosis M. gilvum (NCTC 10742). (2) (Fig. 1). The oligonucleotides were synthesized by using M. chitae (NCTC 10485). a model 381A DNA synthesizer (Applied Biosystems, Foster M. aurum. City, Calif.) according to the instructions of the manufac- M. thermoresistibile (NCTC 10409). turer. The oligonucleotides were purified by fast-protein M. diernhoferi (ATCC 19340). liquid chromatography on a Mono-Q column (Pharmacia M. rhodesiae (ATCC 27024) . M. gadium. Fine Chemicals, Uppsala, Sweden). PCR. A PCR in which we used heat-stable DNA polymer- Nocardia asteroides. ase from Thermus aquaticus (AmpliTaq; Perkin Elmer Ce- N. brasiliensis. tus, Norwalk, Conn.) was performed by using an Intelligent N. farcinica. heating block (Hybaid Ltd., Teddington, United Kingdom) N. otitidiscaviarum. essentially as described by Saiki et al. (16); 100 pul of a reaction mixture containing 50 mM KCl, 10 mM Tris hydro- Staphylococcus aureus . chloride (pH 8.3), 1.5 mM MgCl2, 100 ,ug of gelatin per ml, Staphylococcus epidermidis. each of the deoxynucleotides (Pharmacia) at a concentration Streptococcus pyogenes. Group G streptococcus. of 300 ,uM, 20 pmol of oligonucleotide MT1, 20 pmol of Human leukocytes. oligonucleotide MT2, and 10 pli of a target DNA solution was overlaid with 100 pul of light mineral oil (Sigma) and dena- a Target DNA was extracted and the PCR was performed as described in tured at 98°C for 10 min. AmpliTaq enzyme (2.5 U) was the legend to Fig. 3. The PCR products were analyzed on 3% agarose gels. added, and the PCR was performed. The thermocycling profile consisted of 34 cycles with denaturation at 92°C for 1 polysaccharides, and the remaining proteins were removed min, annealing at 58°C for 1 min, and polymerization at 72°C by precipitation with cetyltrimethyl-ammonium bromide, for 1 min. which was followed by extraction with chloroform-isoamyl Analysis of DNA.