Direct Detection of Mycobacterium tuberculosis in Sputum by ...

JOURNAL OF CLINICAL MICROBIOLOGY, July 1993,

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Vol. 31, No. 7

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0095-1137/93/071777-06$02.00/0 Copyright © 1993, American Society for Microbiology

Direct Detection of Mycobacterium tuberculosis in Sputum by Polymerase Chain Reaction and DNA Hybridization FREDERICK S. NOLTE,l 2,3* BEVERLY METCHOCK,3'4 JOHN E. McGOWAN, JR.,3'4 ALISE EDWARDS,2 OGI OKWUMABUA,2'3 CATHY THURMOND,2 P. SHAWN MITCHELL,2 BONNIE PLIKAYTIS,5 AND THOMAS SHINNICK5 Emory University Hospital, 1 The Emory Clinic, 2 and Department of Pathology and Laboratory Medicine, Emory University School of Medicine,3 Atlanta, Georgia 30322; Grady Memorial Hospital, Atlanta, Georgia 303554; and Hansen's Disease Laboratory, Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303335 Received 8 January 1993/Accepted 4 April 1993

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100°C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%o). This PCR assay incorporates several innovations that make application of this new technology feasible in clinical microbiology laboratories.

(strand displacement amplification) for M. tuberculosis. Many of these procedures used cumbersome sample preparation methods, used radioisotopic methods for amplicon detection, or failed to assess the efficacy of each reaction. None have been developed in a format that would limit the occurrence of false-positive results because of the carryover of PCR products from previous amplification reactions. These factors present substantial obstacles to the application of this new technology in clinical laboratories. We describe a PCR-based assay for M. tuberculosis that incorporates a simple sample preparation protocol, an internal positive control, chemiluminescence detection of hybridization, and a protocol to limit false-positive results because of amplicon contamination. The assay detects a 317-bp target located within IS6110, an insertion sequence that is repeated 10 to 12 times in the chromosomes of M. tuberculosis complex strains (23). We compared the results of the PCR assay with conventional methods for the detection of M. tuberculosis in 313 sputum specimens. (This work was presented in part at the 92nd General Meeting of the American Society for Microbiology, New Orleans, La., 26 to 30 May 1992 [14a].)

A recent World Health Organization survey estimated that in 1990 there were 8 million new cases of tuberculosis worldwide, with 2.9 million deaths directly attributable to the disease (10). Renewed interest in tuberculosis in the United States was prompted by recent increases in the number of new cases and by outbreaks of multiple-drugresistant strains. The lack of simple, rapid, and reliable tests that can specifically detect Mycobacterium tuberculosis in clinical specimens poses enormous problems for both individual patient management and implementation of appropriate infection control and public health measures. The polymerase chain reaction (PCR) is an in vitro method for the specific amplification of target nucleic acid sequences through repeated cycles of thermal denaturation, oligonucleotide primer annealing, and primer extension by a thermostable DNA polymerase (17). PCR and similar nucleic acid amplification strategies are expected to have a significant impact on infectious disease diagnosis, especially for infections involving agents that are difficult, slow, or impossible to culture in vitro. Because of the limitations of the currently available diagnostic tests for tuberculosis, several groups of investigators have developed PCR assays for the direct detection and identification of M. tuberculosis in clinical specimens (1, 4, 6-8, 11, 14, 15, 18, 19, 21). Walker et al. (25) recently described an isothermal DNA amplification assay *

MATERIALS AND METHODS Clinical specimens. Sputum specimens were obtained from the Clinical Microbiology Laboratory of Grady Memorial Hospital, a large county hospital serving an urban population

Corresponding author. 1777

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J. CLIN. MICROBIOL. BamHI 1 Pstd

SmaI

(25)

(280)

pBluescript

PsdI

(617)

TB41

TB43

FIG. 1. Diagram of the control DNA, pTBC-1, used to detect the presence of amplification inhibitors. It contains sequences complementary to the primers TB41 and TB43 separated by a 617-bp fragment of the mycobacterial genome. Numbers in parentheses are in base pairs.

with a high prevalence of tuberculosis. Specimens were digested and decontaminated by the N-acetyl-L-cysteine-2% NaOH method and were concentrated by centrifugation at 3,000 x g for 15 min (9). The sediment was resuspended in approximately 2.0 ml of supernatant. Approximately 1.0 ml of resuspended sediment was used to prepare smears for fluorochrome staining and to inoculate two LowensteinJensen slants and one Middlebrook 7H11 agar plate. Mycobacterial cultures were incubated for a total of 6 weeks. Smear results were quantified by accepted methods (9). The remaining resuspended sediment was processed for PCR. M. tuberculosis and Mycobacterium avium complex strains were identified with commercially available nucleic acid probes (Gen-Probe, San Diego, Calif.), and other species of mycobacteria were identified by conventional methods (9). Bacterial strains and plasmids. M. tuberculosis H37Rv was used as a positive control for the PCR assay. Control DNA that produces a 632-bp PCR product with primers TB41 and TB43, yet that does not hybridize with the TB42 probe, was used as an internal control to detect the presence of amplification inhibitors in clinical specimens (Fig. 1). This control DNA was constructed by using two oligonucleotides, TB41-A, which contained the sequence of TB41 with a BamHI restriction site at the 5' end and a PstI restriction site at the 3' end, and TB43-A, which contained the sequence of TB43 with an EcoRI restriction site at the 5' end and a PstI restriction site at the 3' end. The two oligonucleotides were annealed at the overlapping PstI site and the recessed ends were filled with the Klenow fragment. This fragment was then digested with BamHI and EcoRI and was ligated into pBluescript II KS digested with BamHI and EcoRI. This construct was then digested with PstI, and the 617-bp fragment of the mycobacterial genome was inserted. This plasmid designated pTBC-1 was digested with KpnI and 0.5 pg was added as an internal control in the PCR. PCR assay. (i) Sample preparation. Approximately 1.0 ml of sputum concentrate was transferred to a microcentrifuge tube and was centrifuged at 12,000 x g for 2 min. The pellet was resuspended in 100 ,ul of sample buffer consisting of 10 mM Tris (pH 8.0), 1 mM EDTA, and 1% Triton X-100 (TET), and the mixture was stored at -20°C. The specimens were thawed and then placed in a heat block at 100°C for 30 min. The lysates were centrifuged for 2 min to pellet debris, and 5 to 10 ,ul of the supernatant was used for the PCR assay. The DNA from specimens with amplification inhibitors was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in distilled water. (ii) Oligonucleotide primers and probes. The target for the PCR assay was IS6110, an insertion sequence-like element found only in M. tuberculosis complex strains. The oligonucleotides TB41 (5'-CCTGCGAGCGTAGGCGTCGG-3'; residues 884 to 865; which is the complement of the previously published sequence [22]) and TB42 (5'-CTCGTCCAGCGC

CGCTTCGG-3'; residues 762 to 781) were used as primers, and the oligonucleotide TB43 (5'-TCAGCCGCGTCCACGC CGCCA-3'; residues 568 to 588) was used as the hybridization probe (the residue numbers are those described by Thierry et al. [22]). TB41 and TB43 were previously described by Plikaytis et al. (16). TB42 was labeled with a biotin-dUTP oligonucleotide 3'-end-labeling kit (Clonetech, Palo Alto, Calif.) or with 32P at the 5' end with a T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, Ind.) according to the manufacturer's instructions. Some samples were also amplified with primers PCO4 and GH20, which are specific for the human ,-globin sequence, to assess the efficacy of the amplification reaction (17). (iii) DNA amplification. PCR with clinical specimens was performed in a total volume of 50 or 100 pl in 10 mM Tris-HCI (pH 8.3), 3.25 mM MgCl2, 50 mM KCI, 0.001% gelatin, 200 ,M (each) dATP, dCTP, and dGTP, 600 p,M dUTP, 0.5 p,M (each) TB41 and TB43, 0.025 U of Thermus aquaticus DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) per ,ll, and 0.01 U of Escherichia coli uracil N-glycosylase (UNG; Perkin-Elmer Cetus) per pl. Preliminary experiments were done with a standard PCR protocol in which dTIP (200 ,uM) was substituted for dUTP, the concentration of MgCl2 was 1.5 mM, and UNG was omitted. The reaction mixtures were overlaid with mineral oil, and those with UNG were held at room temperature for 10 min to allow the enzyme to degrade any contaminating dU-containing PCR products from previous amplifications. The tubes were then subjected to 30 to 40 thermal cycles in a programmable heat block (Perkin-Elmer Cetus) by using a two-step cycle of 1 min and 15 s at 94°C (denaturation) and then 3 min at 68°C (primer annealing and extension). PCR products were stored at 72 or -20°C to prevent degradation by any residual or reactivated UNG. In addition to the dUTP-UNG protocol, we used the practices recommended by Kwok and Higuchi (12) to limit false-positive PCR results. (iv) Product detection. Amplified DNA (25 ,ul) was electrophoresed in 2.0% agarose gels, and the 317-bp product was visualized by staining with ethidium bromide and UV light transillumination. The gel was denatured and neutralized, and the DNA was transferred to Duralon membranes (Stratagene, La Jolla, Calif.) by Southern blotting (20). The membranes were placed in a prehybridization solution (3 x SSPE [lx SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA; pH 7.7], 5 x Denhardt's solution, 30% formamide, 0.5% sodium dodecyl sulfate [SDS], 1 mM EDTA, and 1 mM Tris-HCl [pH 8.0]) for 15 min at 42°C. A total of 5.0 pmol of biotin-labeled TB42 probe per ml was added, and hybridization occurred at 42°C for 1 h. After hybridization, the membranes were washed sequentially at ambient temperature for 5 min in 2x SSC (lx SSC is 0.15 M NaCl plus 0.15 M sodium citrate), at 60°C for 30 min in 2x SSC with 0.1% SDS, and at ambient temperature for 15 min in 0.16x SSC

DETECTION OF M. TUBERCULOSIS BY PCR

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FIG. 2. Autoradiograph of a Southern blot of PCR products obtained from serial 10-fold dilutions of a culture-positive sputum specimen probed with TB42. Aliquots of each dilution were also cultured onto Middlebrook 7H11 agar. The limit of detection by culture and PCR were the same at the 10-' dilution (lane 7). Lane 1, reagent blank; lanes 2 to 11, 100 to 10'- dilutions of a culturepositive sputum, respectively; lanes 12 and 13, positive controls. The arrow indicates the position of the 317-bp amplicon.

with 0.1% SDS. A chemiluminescent Southern blot procedure was used to detect hybridized probe (Southern-Light; Tropix, Inc., Bedford, Mass.). Results were imaged on X-ray film after 30 to 60 min of exposure. (v) Controls. A suspension of 100 to 1,000 CFU of M. tuberculosis H37Rv and a reagent blank (no target DNA) were included in each run. A second amplification reaction was performed for each specimen that was negative for the 317-bp product to detect the presence of PCR inhibitors. Initially, specimens were spiked with 0.1 ng of human genomic DNA and a segment of the 0-globin region was amplified with the primers PCO4 and GH20. Later in the study, specimens were spiked with 0.5 ng of pTBC-1 and a 678-bp sequence was amplified with TB41 and TB43. Those specimens from which the internal control product was not detected by gel analysis were considered inhibitory. RESULTS The analytical sensitivity of the standard PCR assay was assessed in several ways. The assay was able to reliably detect 10 fg of purified M. tuberculosis chromosomal DNA diluted in 0.1 ng of human genomic DNA after Southern blotting and hybridization. This corresponds to approximately two mycobacteria. More meaningful measures of the assay's sensitivity were obtained by terminal dilution experiments performed with suspensions of M. tuberculosis H37Rv and positive sputum specimens. Serial ten-fold dilutions of an H37Rv suspension and two positive sputum concentrates were prepared in TET, and the limit of detection by PCR and culture was compared for these dilution series. The culture involved plating 100 pl of each dilution on Middlebrook 7H11 agar and counting the colonies after 6 weeks of incubation. The sample volume used in the PCR assay was 10 ,ul. The limit of detection by culture and PCR was equivalent (10-' dilution) for both the organism suspension and the positive sputum specimens. Figure 2 shows the results of the terminal dilution experiment with a positive sputum specimen. The hybridization product below the 317-bp amplicon resulted from single-stranded DNA produced by asymmetric amplification when one of the primers was depleted. A crude assessment of the efficiency of the lysis of mycobacteria by our PCR sample preparation method was made by microscopy. Examination of Kinyoun-stained smears prepared from heated and unheated suspensions of M. tuberculosis H37Rv revealed that heat treatment disrupted approximately 90% of the cells. In addition, the PCR sample preparation method sterilized suspensions containing 106 CFU of strain H37Rv per ml.

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FIG. 3. Autoradiograph of a Southern blot of PCR products obtained from serial 10-fold dilutions of a suspension of M. tuberculosis H37Rv (= 10' CFU/ml) probed with TB42. (A) Lanes 1 to 6, 100 to 10'- dilutions of H37Rv amplified by the standard PCR protocol, respectively. (B) Lanes 1 to 6, the same dilutions of H37Rv as in panel A amplified by the dUTP-UNG PCR protocol. The limit of detection was the 10-3 dilution by both PCR protocols (lanes 4).

Although the IS6110 sequence has been well characterized and found only in species belonging to the M. tuberculosis complex (5, 22), we amplified DNA from stock mycobacterial strains of M. bovis BCG, M. africanum, M. avium complex, M. kansasii, M. chelonae, M. fortuitum, and M. gordonae. As expected, only those species belonging to the M. tuberculosis complex showed the specific 317-bp product after amplification (data not shown). Use of the dUTP-UNG PCR protocol required that the concentration of dUTP be increased threefold over the concentrations of the other deoxynucleotide triphosphates and that the MgCl2 concentration be increased to 3.25 mM for efficient amplification. With these modifications we found no difference in the limits of detection between standard and dUTP-UNG PCR protocols in terminal dilution experiments with purified target DNA and strain H37Rv lysates. Figure 3 shows the results of a comparison of the two protocols with dilutions of an H37Rv lysate. We conducted intentional contamination experiments to test the ability of the dUTP-UNG protocol to degrade or eliminate large numbers of PCR products that were carried over. Figure 4 shows the results of one such experiment in which two series of reaction tubes were "contaminated" 1

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5

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I1

4 FIG. 4. Autoradiograph of a Southern blot of PCR products obtained from serial 10-fold dilutions of dU-containing amplicon from a previous amplification reamplified by the dUTP-UNG protocol (lanes 2 to 5) and the standard protocol (lanes 8 to 11). Lane 1, reagent blank; lanes 2 to 5, 10-2 to 10' dilutions of the dUcontaining amplicon reamplified by the dUTP-UNG protocol, respectively; lane 6, native template amplified by the dUTP-UNG protocol; lane 7, reagent blank; lanes 8 to 11, 10-2 to 10' dilutions of dUTP-containing amplicon reamplified by the standard PCR protocol, respectively. The dUTP-UNG protocol prevented reamplification of the dU-containing amplicon at the dilutions tested.

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J. CLIN. MICROBIOL. TABLE 1. Correlation of acid-fast bacillus smear quantitation and PCR results for 124 sputum specimens containing M. tuberculosis No. (%) of PCR positive

Smear quantitation (no. of specimens)

4+ 3+ 2+ 1+ ,

4

6

8

9

10

Il 1X

(75) ............................................... (12) ............................................... (17) ......................................... (6) ..........

...............................

Negative (14) .........................................

13

74 (99) 11 (92) 16 (94) 4 (67) 8 (57)

B

FIG. 5. Electrophoretic separation of DNA amplified from

spu-

tum specimens by PCR. One-half (25 pl) of the reaction mixture was

electrophoresed in a 2.0% agarose gel and stained with ethidium bromide (A). The DNA was transferred to a membrane, which was probed with a biotin-labeled oligonucleotide, TB42 (B). A chemiluminescent reporter molecule was used to image the hybridized probe on X-ray film after 30 min of exposure. Lanes 1, molecular size standards; lanes 2, reagent blank; lanes 3, positive control containing M. tuberculosis H37Rv and pTBC-1; lanes 4 to 8, culture-positive sputum specimens (M. tuberculosis); lanes 9 to 13, culture-negative sputum specimens spiked with pTBC-1 (internal control). The arrows indicate the positions of the 317-bp target and the 632-bp internal control amplicons.

with dilutions of dU-containing product. The initial reaction mixture contained approximately 1,000 copies of native M. tuberculosis template DNA. One series was amplified by the standard protocol and the other was amplified by the dUTPUNG protocol. The dUTP-UNG protocol prevented reamplification of the dU-containing product from a previous reaction at all dilutions tested. An example of an ethidium bromide-stained gel containing amplified DNA from 10 sputum specimens is shown in Fig. 5A. Five of the specimens were culture positive for M. tuberculosis and were positive for the IS6110 product (317 bp) by PCR. Figure 5A also shows the 632-bp product of the internal control used to assess the efficacy of the PCR for the five specimens that were negative for the 317-bp product. Chemiluminescence imaging of the Southern blot prepared from the gel shown in Fig. 5A and probed with biotin-labeled TB42 is shown in Fig. SB. The results of the PCR assay, based on hybridization detection, and the culture results for detection of M. tuberculosis in 313 sputum specimens are as follows. Of 123 specimens that were culture positive for M. tuberculosis, 112 (91%) were positive for M. tuberculosis by PCR; of 17 specimens that were culture-positive for mycobacteria other than M. tuberculosis, none were positive by PCR; of 173 specimens that were culture negative for mycobacteria, 1 (0.58%) was positive by PCR; this was an acid-fast bacillus smear-positive specimen that was obtained from a patient receiving treatment for culture-proven tuberculosis. The PCR assay detected M. tuberculosis in 91% of the culturepositive specimens. The smear quantitation and correlation with the PCR results for the culture-positive sputum specimens are shown in Table 1. The PCR assay detected 105 of 110 (95%) smear-positive specimens and 8 of 14 (57%) smear-negative specimens that contained M. tuberculosis. Table 2 compares the results of gel analysis and hybrid-

ization for detection of the M. tuberculosis amplicon. PCR products with sizes similar to that of the specific 317-bp product and that did not hybridize to TB42 were amplified in five specimens. These specimens would have been interpreted as positive if hybridization was not done. Southern blotting and hybridization with TB42 also detected eight additional true-positive specimens. The hybridization assay substantially improved the sensitivity and specificity of the assay.

We found that 17% of the specimens contained PCR inhibitors of the 0-globin internal control and that 10% contained inhibitors of the pTBC-1 internal control. DNAs from these inhibitory samples were extracted with phenolchloroform, precipitated with ethanol, and retested. These additional sample processing steps removed the inhibitors in all cases. PCR-positive results for five (4.4%) specimens were obtained after these additional specimen processing steps.

DISCUSSION The increasing number of new cases of tuberculosis and the rising numbers of multiple-drug-resistant M. tuberculosis have prompted interest in the development of rapid diagnostic tests for tuberculosis. Rapid assays based on nucleic acid amplification techniques such as PCR have great potential, but many practical challenges must be met before these assays can be brought into routine clinical use. The PCR assay that we developed incorporated several innovations that make the application of this new technology in clinical laboratories feasible. These innovations include a simple sample preparation method, the use of an internal positive control, chemiluminescence detection of hybridization, and a means of preventing false-positive results because of amplicon carryover. A variety of sample preparation methods have been described for PCR-based assays for mycobacteria. Most have included detergents and proteolytic enzymes to disrupt the mycobacteria and release the DNA and organic solvents or nucleic acid capture reagents to purify the target DNA. The TABLE 2. Gel analysis versus hybridization for detection of the M. tuberculosis amplicon Test method

Gel analysis Hybridization

No. of specimens: True False

Percent

positive

positive

Sensitivity

Specificity

105 113

5a

0

85 91

97 100

a Samples with DNA bands of approximately 317 bp that did not hybridize with the TB42 probe.

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sample preparation method that we used was first described by Sritharan and Barker (21). They found that the TETboiling method lysed 95% of mycobacteria and permitted detection of fewer than 10 mycobacteria in a sample by their PCR assay. The results that we obtained after using this sample preparation method were similar. This method is technically simple, requires few manipulations, and sterilizes the sample. It compared favorably with other methods of sample preparation evaluated by Sritharan and Barker (21) and with the methods described by Eisenach et al. (6) and Brisson-Noel et al. (1), which we also evaluated in our laboratory (data not shown). Buck et al. (2) recently compared several simple methods of sample preparation for an M. tuberculosis PCR assay and found that boiling with nonionic detergents, including Triton X-100, was a relatively ineffective means of sample preparation. The reason for the poor results obtained by that sample preparation method is not clear since neither the efficiency of lysis nor the presence of PCR inhibitors was assessed for samples prepared by the various methods that they investigated. The presence of PCR inhibitors in clinical specimens is a major obstacle to the routine application of PCR in clinical laboratories. PCR assays should include internal controls in order to assess the efficacy of each amplification reaction and to ensure that the sample is free of interfering substances. The use of internal controls will identify those samples that are inappropriate for PCR or that require further manipulation to remove inhibitors, and their use will ultimately increase confidence in the reliability of negative results. We used two internal controls during the course of the present study, initially, human placental DNA and, later, pTBC-1. With these internal controls we found complete inhibition of the amplification of the control target DNA sequences in 13.6% of the sputum samples. The DNAs from these samples were extracted with phenol-chloroform and were precipitated with ethanol. Although these additional sample preparation steps effectively removed the inhibitors, concern about the ability of this procedure to recover small quantities of target DNA led us to purify DNA only from those negative samples that failed to amplify the internal control. pTBC-1 was developed so that both the specific target and the internal control could be amplified in the same reaction tube, with the same primer pair, and with the same thermal cycle parameters. Unfortunately, the pTBC-1 sequence was preferentially amplified and the analytical sensitivity of the assay for the IS6110 target was reduced 10- to 100-fold in the presence of the internal control. Attempts to restore the analytical sensitivity of the assay in the presence of the internal control by adjusting the concentrations of the various components of the PCR mixture were unsuccessful. As a result, a second amplification reaction was necessary to assess the presence of PCR inhibitors. Eisenach et al. (6) constructed a similar internal control for their PCR assay; that internal control consisted of two 20-bp primer sequences separated by 550 bp of DNA isolated from Salmonella typhimurium. Eisenach et al. (6) showed that the presence of the internal control did not diminish the amplification of target DNA. The DNA that separated the primer sequences in pTBC-1 was isolated from M. tuberculosis rather than S. typhimurium. The difference in the spacer DNA used to construct the two internal controls may account for the different effects of the control DNA on the amplification of the specific target sequences. Another problem facing the application of PCR in routine diagnostic laboratories is the occurrence of false-positive

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reactions caused by contaminating amplicons. Each PCR tube may contain up to 1012 copies of an amplicon, and the smallest aerosolized droplet may contain as many as 105 copies of that amplicon. Since each amplicon can serve as a template for subsequent PCRs, considerable efforts have been devoted to devising ways to limit amplicon carryover. These include the specific laboratory practices and procedures described by Kwok and Higuchi (12) as well as recently described amplicon sterilization methods (3, 13). The dUTP-UNG protocol used here effectively sterilized the IS6110 amplicon, with minimal effects on the analytical sensitivity of the assay once the concentrations of dUTP and MgCl2 were adjusted. No false-positive PCR assay results were encountered for the 313 sputum specimens evaluated. By reducing the number of false-positive results because of amplicon carryover, effective amplicon sterilization methods can increase the level of confidence in positive results. In addition, these methods may have the added practical benefit of reducing the amount of laboratory space required to perform this type of analysis. However, until more experience is gained with these methods, the physical separation of pre- and postamplification procedures is prudent. The PCR assay described here detected 95% of smearpositive and 57% of smear-negative sputum specimens that contained M. tuberculosis. Shawar et al. (18), using a sample preparation and amplification protocol similar to that described here, detected 90% of smear-positive and 53% of smear-negative specimens. Although PCR-based diagnostic tests should have the greatest impact in detecting paucibacillary disease, few studies have included large numbers of smear-negative, culture-positive sputum specimens in the evaluation of PCR-based assays. During the time that specimens were selected for use in the present study, approximately 30% of the culture-positive sputa were fluorochrome smear negative. In practice, the overall diagnostic sensitivity of the PCR assay would be lower than the 91% reported here, since only 14% of the culture-positive sputa included in the study were smear negative. Recently, Victor et al. (24) reported that nine of nine smear-negative, culture-positive specimens were detected by PCR, but only after two 30-cycle amplifications. For sputum specimens containing few mycobacteria, the sample size becomes a major limitation for PCR-based assays. Culture can easily accommodate 0.5 to 1.0 ml of sputum concentrate, whereas the sample volumes for PCR-based tests are usually 10 to 20 ,ul. The volume advantage of culture may partially explain the relatively poor performance of PCR with sputum specimens containing few mycobacteria.

The hybridization assay that we used for the detection and confirmation of the IS6110 amplicon increased both the sensitivity and the specificity of the PCR assay. The falsepositive results obtained on the basis of interpretation of the ethidium bromide-stained agarose gels were due to the presence of PCR products in five samples that were similar in size to the specific 317-bp product. These products failed to hybridize to our specific probe. We used chemiluminescent probes rather than radioisotopes to detect the IS6110 amplicon in Southern blots. The advantages of the chemiluminescence system include longer probe shelf-life, shorter film exposure times, and reduced cost because of the elimination of institutional charges associated with the acquisition, monitoring, and disposal of radioisotopes. Kolk et al. (11) used a digoxigenin-labeled probe, rabbit anti-dioxigeninalkaline phosphatase, and colorimetric methods to detect hybridized DNA in a PCR-based assay for M. tuberculosis.

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Shawar et al. (18) used both digoxigenin- and alkaline phosphatase-labeled probes to detect an IS6110 amplicon. These nonradioactive detection systems are practical alternatives to the use of 32P-labeled probes for clinical laboratories. We developed a PCR-based assay for the rapid, direct detection and identification of M. tuberculosis in clinical specimens. The assay incorporates several innovations that make the application of this new technology feasible in clinical microbiology laboratories. One technologist can process up to 30 specimens per day, and the assay requires only 14 h to complete. We are evaluating the test performance characteristics, practicality, and cost-effectiveness of the PCR assay relative to those of conventional tests for the diagnosis of pulmonary tuberculosis in a large, prospective clinical trial that includes approximately 2,400 consecutive specimens. ACKNOWLEDGMENT This work was supported by grant U52/CCU406008 from the U.S. Public Health Service (Centers for Disease Control and Prevention). REFERENCES 1. Brisson-Noel, A., C. Aznar, C. Chureau, S. Nguyen, C. Pierre, M. Bartoli, R. Bonette, G. Pialoux, B. Gicquel, and G. Garrigue. 1991. Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet 338:364-366. 2. Buck, G. E., L. C. O'Hara, and J. T. Summersgill. 1992. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J. Clin. Microbiol. 30:1331-1334. 3. Cimino, G. D., K. C. Metchette, J. W. Tessman, J. E. Hearst, and S. T. Isaacs. 1990. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res. 19:99-107. 4. DeWit, D., L. Steyn, S. Shoemaker, and M. Sogin. 1990. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J. Clin. Microbiol. 18:2437-2441. 5. Eisenach, K., J. T. Crawford, and J. H. Bates. 1988. Repetitive DNA sequences as probes for Mycobacterium tuberculosis. J. Clin. Microbiol. 26:2240-2245. 6. Eisenach, K. D., M. D. Sifford, M. D. Cave, J. H. Bates, and J. T. Crawford. 1991. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am. Rev. Respir. Dis. 144:1160-1163. 7. Hance, A. J., B. Grandchamp, V. Levy-Frebault, D. Lecossier, J. Rauzier, D. Bocart, and B. Gicquel. 1989. Detection and identification of mycobacteria by amplification by mycobacterial DNA. Mol. Microbiol. 3:843-849. 8. Hermans, P. W., R. J. Schuitema, D. V. Soolingen, C. P. H. J. Verstynen, E. M. Bik, J. E. R. Thole, A. H. J. Kolk, and J. D. A. van Embden. 1990. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J. Clin. Microbiol. 28:1204-1213. 9. Kent, P. T., and G. P. Kubica. 1985. Public health mycobacteriology. A guide for the level III laboratory. Centers for Disease Control, Atlanta. 10. Kochi, A. 1991. The global tuberculosis situation and the new

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