May 11, 1992 - Braun, M. M., B. I. Truman, B. Maguire, G. T. DiFerdinando,. G. Wormser, R. Broaddus, ... Andrew, and G. J. Boulnois. 1991. A DNA primer probe ...
Vol. 30, No. 8
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2173-2176 0095-1137/92/082173-04$02.00/0 Copyright C) 1992, American Society for Microbiology
Characterization of a DNA Probe for Detection of Mycobacterium tuberculosis Complex in Clinical Samples by Polymerase Chain Reaction MANUEL ALTAMIRANO,1* MICHAEL T. KELLY,1'2 ALFRED WONG,' ELAINE T. BESSUILLE,2 WILLIAM A. BLACK,1'2 AND JOHN A. SMITH" 2 Division of Medical Microbiology, Department of Pathology, University of British Columbia, Vancouver, British Columbia V5Z 1M9, 1 and Bnitish Columbia Centre for Disease Control, Vancouver, British Columbia V5Z 1L8,2 Canada Received 17 January 1992/Accepted 11 May 1992
We cloned and sequenced a DNA fragment from Mycobacterium tuberculosis for use in the identification of members of the M. tuberculosis complex. The DNA probe for culture confirmation had a sensitivity and a specificity of 100%o. By using primers developed from this probe, the polymerase chain reaction detected 20 mycobacteria by ethidium bromide staining. This polymerase chain reaction system demonstrated 98% sensitivity and 100%o specificity for detection of the M. tuberculosis complex in 200 sputum specimens.
5 min at room temperature, and extracted with an equal volume of chloroform-isoamyl alcohol (24:1). The phases were then separated by centrifugation in a microcentrifuge for 15 min. The supernatant was recovered and precipitated with ethanol, and the pellet was resuspended in 25 ,ul of water. The DNA concentration was determined by using the TKO 100 minifluorometer (Hoefer Scientific Instruments, San Francisco, Calif.) with the fluorometric dye Hoechst 33258 (8). One liter of a 6-week-old culture of the well-characterized M. tuberculosis H37Rv strain TMC 102 was grown in Middlebrook 7H9 broth supplemented with oleic acid, albumin, dextrose, and catalase (OADC; Difco, Gaithersburg, Md.). Cycloserine (1 mg/ml) was added, and 24 h later, the culture was heated at 70°C for 15 min and harvested by centrifugation for DNA extraction and purification (16). EcoRI-digested M. tuberculosis DNA was cloned in plasmid pT7T3 18U by using Escherichia coli MN522 as a host; positive clones were selected by hybridization by established procedures (19). The double-stranded DNA sequence was determined by using the Automatic Laser Fluorescent DNA sequencer from Pharmacia LKB (Uppsala, Sweden) and the AutoRead Sequencing Kit (Pharmacia) according to the manufacturer's instructions. PCR amplification was carried out in 50-il-volume reactions containing (final concentrations) 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, 200 ,uM (each) the four deoxyribonucleoside triphosphates, 320 nM each primer (primer 1, 5'CAAGGCT7CAATrCCGGTGATGCC-3'; primer 2, 5'-TG GTCCGGTTCATACTCGGGCTGG-3'), template DNA in 10 ,u, and 1.5 U of recombinant Ampli-Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) covered with 25 ,ul of mineral oil. A Perkin-Elmer Cetus DNA thermal cycler was used for 35 cycles of denaturing at 94°C for 1.5 min, annealing at 70°C for 1 min, and extension at 72°C for 1 min. Ten percent of the reaction was analyzed by electrophoresis in a 2% agarose gel by using a 123-bp DNA ladder (Bethesda Research Laboratories) as a marker. Gels were stained with ethidium bromide and were photographed under a 300-nm UV transilluminator (Fotodyne, New Berlin, Wis.). Samples were considered to be positive for the M. tuberculosis complex when a single band of 285 bp was present. The gel
Tuberculosis, which continues to be an important disease worldwide, has recently seen a resurgence in North America largely because of the AIDS epidemic and the influx of infected immigrants (3, 5, 7). Despite recent advances such as the radiometric detection of Mycobacterium tuberculosis (14), the laboratory diagnosis of tuberculosis is still hampered by the slow growth of the organism, and new methods are needed for the rapid detection of M. tuberculosis in clinical specimens. Nucleic acid probes have been used for the identification of mycobacterial cultures (2, 16), but they lack sensitivity for mycobacterial detection in clinical specimens. Amplification of mycobacterial DNA sequences by the polymerase chain reaction (PCR) has shown more promise for mycobacterial detection in clinical specimens (4). In the present report we describe the development of a PCR system for the rapid and accurate detection of M. tuberculosis in sputum specimens. The following mycobacterial type strains were obtained from the American Type Culture Collection (ATCC; Rockville, Md.): M. tuberculosis H37Rv (ATCC 27294), M. africanum ATCC 25420, M. avium ATCC 5714, M. bovis ATCC 19274, M. chelonae ATCC 14472, M. intracellulare ATCC 13950, M. kansasii ATCC 12478, M. marinum ATCC 927, M. microti ATCC 19422, M. paratuberculosis ATCC 19698, M. simiae ATCC 25275, M. szulgai ATCC 35799, and M. terrae ATCC 15755. Clinical mycobacterial isolates (Table 1) and 300 sputum samples were obtained from the British Columbia Centre for Disease Control. Forty-two isolates of bacteria other than mycobacteria (BOTM) were obtained from the Division of Medical Microbiology, Vancouver General Hospital. Sputum samples were liquefied, decontaminated, and concentrated by standard methods (18). Concentrated sputum or samples from pure cultures were resuspended in 300 pl of TE buffer (10 mM Tris-HCl, 1 mM EDTA [Sigma Chemical Co., St. Louis, Mo.] [pH 8.0]), sodium dodecyl sulfate (concentration, 1% [wt/vol]; Sigma), and lysozyme (1.6 mg) were added, and the reaction mix was incubated at 37°C for 20 min. Samples were adjusted to a final concentration of 1 M sodium perchlorate, incubated for *
Corresponding author. 2173
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J. CLIN. MICROBIOL.
NOTES TABLE 1. Mycobacterial isolates used in this study
Species
or group
M. tuberculosisa......................................... M. avium-M. intracellulare ................................... M. chelonae ......................................... M. paratuberculosis ......................................... M. fortuitum ......................................... M. kansasii ......................................... M. marinum ......................................... M. terrae ......................................... M. szulgai......................................... M. xenopi .............. ........................... M. scrofulaceum ......................................... M. simiae ......................................... M. africanuma ......................................... M. bovisa ......................................... M. microtia ......................................... Scotochromogenic group ...................................... Nonphotochromogenic group ................................ MAIS group ................. ........................
Total ......................................... a Members of the M. tuberculosis complex.
No. of isolates tested
A -
100 41 21 8 7 3 2 2 2 1 1 1 1 1 1 10 5 1
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was vacuum blotted onto a nylon membrane for hybridiza-
tion analysis. Nucleotide sequence accession number. The DNA sequence of clone ASK58 (959 bp) has been deposited in GenBank and has the accession number M75726. Ten thousand recombinants were obtained from the cloning experiments. After colony hybridization, clone ASK58, which gave a strong signal for M. tuberculosis, was chosen, and the specificity of the 959-bp insert, which was used as a DNA probe, was tested by using the mycobacterial collection. The DNA probe hybridized only to M. tuberculosis as a single band. The DNA sequence of clone ASK58 was determined and was found to have had a G+C content of 69.4%. A search for homology was made in the GenBank release number 70 from January 1992, but no similar sequences were found. The insert of clone ASK58 was restricted with BamHI, generating three fragments of 162, 318, and 480 bp. The 318-bp fragment was subcloned into plasmid pT7T3 18U (subclone ASK58-4). The insert that was retrieved was used as a DNA probe for subsequent experiments. DNAs from 223 mycobacterial isolates were analyzed with the DNA probe. All 103 samples of the M. tuberculosis complex gave a positive signal, while the other 105 mycobacteria other than M. tuberculosis (MOTT) samples (Fig. 1; Table 1) and 15 BOTM cultures did not hybridize (specificity and sensitivity, 100%). One hundred sputum samples were examined by culture and DNA hybridization. Culture yielded five strains of M. tuberculosis, three strains of M. avium-M. intracellulare, and one Runyon group III mycobacterium; the remaining 91 samples were culture negative. Of the five samples that grew M. tuberculosis, four were detected with the probe and one gave a false-negative result. The MOTT samples did not generate a signal. The limit of detection was 1 ng, which corresponds to 2 x 105 mycobacteria on the basis of an estimated genome molecular weight of 3.0 x 109 (15). Serial dilutions from M. tuberculosis DNA (Fig. 2) and an M. tuberculosis colony (Fig. 3) were amplified by PCR. The limit of detection by using ethidium bromide staining was 100
FIG. 1. Culture confirmation with the DNA probe from M. tuberculosis. (A) Representative slot blot hybridization from 103 culture samples of M. tuberculosis. Fifty-three samples are shown; in all of them there is a hybridization signal. (B) Representative autoradiography from 113 samples of MOTT. Fifty-one slots are shown. Only the slot loaded with DNA from M. tuberculosis gave a positive signal. The MOTT samples are M. chelonae (n = 19), M. avium-M. intracellulare (n = 19), M. fortuitum (n = 3), M. terrae (n = 2), M. scotochromogenic (n = 2), M. marinum (n = 2), M. kansasii (n = 1), M. simiae (n = 1), M. szulgai (n = 1), and nonphotochromogenic mycobacterium (n = 1).
fg and 20 mycobacteria. The limit of detection by autoradiography was 5 fg and one mycobacterium. To evaluate the specificity of the method, DNAs from 103 cultures of the M. tuberculosis complex, 105 MOTT (Table 1), and 27 BOTM (Table 2) were analyzed by PCR. Only the 103 samples of the M. tuberculosis complex were positive. Two hundred sputum samples from patients suspected of having a mycobacterial infection were analyzed by culture A
B
R1 i'"'F3 4 RM-
2
3
4
5
6
7
8
FIG. 2. Limit of detection of PCR with M. tuberculosis DNA by ethidium bromide staining (A) and autoradiography (B). Lanes: 1, 0 fg; 2, 1 fg; 3, 5 fg; 4, 10 fg; 5, 100 fg; 6, 1 pg; 7, 10 pg; 8, 100 pg; R, reactive control; C, M. tuberculosis control; M, 123-bp DNA marker ladder. The limit of detection was 100 fg (A) and 5 fg (B).
NOTES
VOL. 30, 1992 A
B 1
2
3
4
6
5
w
7
8
9
S
FIG. 3. Limit of detection by PCR of DNA extracted from 10-fold dilutions of cultured M. tuberculosis. The numbers of mycobacteria were 2 x 106 (lane 1), 2 x 105 (lane 2), 2 x 104 (lane 3), 2 x 103 (lane 4), 2 x 102 (lane 5), 20 (lane 6), 2 (lane 7), 1 (lane 8), and 0 (lane 9). The limit of detection by ethidium bromide staining was 20 mycobacteria (A), and by Southern blot hybridization it was 1 mycobacterium (B).
and PCR. Of 44 samples that were culture positive for M. tuberculosis, 43 were positive by PCR, while one of the samples was a false negative (sensitivity, 98%). The remaining 156 samples (50 MOTI and 106 culture negative) were negative by PCR (specificity, 100%). Current routine diagnostic methods for tuberculosis are slow and inefficient. This impedes the control of the disease by public health officials and potentially compromises the treatment of the patient. Improved diagnostic methods were recommended by the Centers for Disease Control Advisory Committee for the Elimination of Tuberculosis (6). Previously published protocols used PCR to detect mycobacterial gene sequences that code for rRNA (1) or proteins such as TABLE 2. Single strains of non-Mycobacterium cultures tested and found negative by PCR Strain
Campylobacter jeujuni Candida sp. Cryptococcus neoformans Diphtheroid Enterococci sp. Enterobacter sp. Escherichia coli Haemophilus influenza Klebsiella pneumoniae Klebsiella sp. Neisseria meningitidis Neisseria sp. Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Salmonella enteritidis Serratia liquefaciens Serratia marcescens Shigella sonnei Staphylococcus aureus Coagulase-negative staphylococci Streptococcus group A Streptococcus pneumoniae Streptococcus sp. Viridans group streptococci Xanthomonas maltophilia Yersinia enterocolitica
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those of 65 kDa (4), 38 kDa (20), 24 kDa (12); proteins MPB64 (13) or MPB70 (9); or insertional elements such as IS6110 (11) and IS986 (12). Other systems use repetitive sequences (10) or specific fragments like EcoRI-BamHI (17). Our primers were derived from a unique, 318-bp cloned fragment of M. tuberculosis DNA that functions as a highly specific probe for the organism. The 285-bp amplification product of our PCR is distinct from any of those described previously. Thirty-five cycles of amplification of the DNA present in 20 mycobacteria (100 fg) were able to generate enough PCR product to be visible in an agarose gel stained with ethidium bromide. If there were fewer than 20 mycobacteria in a clinical sample, it was necessary to use a radioactive probe for autoradiography, and the limit of detection was one to two mycobacteria (5 to 10 fg). The system is thus both sensitive and specific for the detection of the organism in sputum samples. Our results confirm that use of the DNA probe for culture confirmation is reliable and illustrate the benefit of using a DNA amplification procedure such as PCR for the accurate detection of the M. tuberculosis complex in clinical samples in 1 or 2 days. This work was supported by grants from Tuberculous and Chest Disabled Veterans' Association, British Columbia, Canada; Woodwards Foundation; the British Columbia Medical Services Foundation, and the Science Council of British Columbia. We thank Ross T. A. MacGillivray (Department of Biochemistry, University of British Columbia) for critically reading the manuscript and for helpful discussions.
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