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Journal of Microbiological Methods 66 (2006) 440 448 www.elsevier.

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Direct and Simultaneous Identification of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium tuberculosis (MTB) by Rapid Multiplex nested PCR-ICT assay
Po-Chi Soo a,1 , Yu-Tze Horng a,c,1 , Po-Ren Hsueh b , Bin-Jon Shen c , Jann-Yuan Wang b , Hui-Hsin Tu a , Jun-Rong Wei a , Shang-Chen Hsieh a , Chien-Chung Huang a , Hsin-Chih Lai a,b,
a

Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC b Department of Laboratory Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC c Tyson Bioresearch Inc., Taiwan, ROC Received 29 November 2005; received in revised form 18 January 2006; accepted 23 January 2006 Available online 3 March 2006

Abstract The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M. tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. The currently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immunochromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application. 2006 Elsevier B.V. All rights reserved.
Keywords: Mycobacterium tuberculosis; Molecular detection; Multiplex nested PCR; Immuno-chromatography test (ICT)

1. Introduction
Corresponding author. Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, No.1., Chan-Der Street, Taipei 100, Taiwan, ROC. Tel.: +886 2 2312 3456x6931; fax: +886 2 2371 1574. E-mail address: hclai@ha.mc.ntu.edu.tw (H.-C. Lai). 0167-7012/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.01.010

Tuberculosis (TB) is the leading cause of human adult death by infectious gents, accounting for approximately two million deaths annually, mainly in the developing countries. Currently, the global number

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of TB cases is rising at a rate of 2% per year (World Health Organization tuberculosis fact sheet; http:// www.stoptb.org/tuberculosis/). While conventional smear microscopy and culture methods are widely used for diagnosis of TB, the former is insensitive (Caws et al., 2000), and the latter takes up to 6 to 8 weeks to provide a result, limiting the value of these methods in aiding diagnosis and immediate decisions on treatment. Using IS6110 and 16S rDNA as detection targets, many commercially available nucleic acid amplification-based detection systems have also been developed as rapid tests for direct identification of Mycobacterium tuberculosis complex (MTBC) from clinical specimens (Gardiner and Beavis, 2000; Soini and Musser, 2001; Woods, 1999). These include PCRbased Amplicor (Roche), ligase chain reaction (Lcx; Abbott Systems), transcription-mediated amplification (TMA; Gen-Probe), strand displacement amplification (BDProbe; Tec-SDA) and the RAPID-BAP-MTB assay (AsiaGen, Taiwan) (Brown et al., 1999; Eing et al., 1998; Hellyer et al., 1996; Piersimoni et al., 1998; Reischl et al., 1998; Wang et al., 2004; Yuen et al., 1997). Variations in the sensitivities and high costs of these tests have hindered these systems from being widely used in TB detection. Although the mycobacteria grouped in the MTBC are closely related based on DNADNA hybridization, multilocus enzyme electrophoresis, and 16S rDNA nuleotide acid sequence level (Boddinghaus et al., 1990; Sreevatsan et al., 1997), MTBC members differ widely in terms of host tropisms, phenotypes, and pathogenicity. It is intriguing that some are exclusively human (M. tuberculosis, Mycobacterium africanum, Mycobacterium canetti) or rodent pathogens (Mycobacterium microti), whereas others either have a wide host spectrum (Mycobacterium bovis) (Brosch et al., 2002) or are used as a vaccine strain (M. bovis BCG). Differentiation of M. tuberculosis (MTB) from the other members of the MTBC is thus necessary for treatment of individual patients and for epidemiological study purposes, especially in areas of the world where tuberculosis has reached epidemic proportions or wherever the transmission of M. bovis between animals or animal products and humans is a problem. While current detection methods do not differentiate MTB from other members of the MTBC (Abe et al., 1993; Alcaide et al., 2000; Katila et al., 2000), recent comparative genomic analyses have provided valuable information on regions of difference (RD) in the chromosome of MTB, and indicated that specific identification of MTB can be achieved by use of these RDs (Parsons et al., 2002). In this study, to rapidly and

specifically identify MTB from sputum samples, the multiplex nested PCR combined with immuno-chromatography test (ICT) assay was developed. Rv3618 DNA fragment which belongs to RD9 (Behr et al., 1999) was selected as a potential target for MTB diagnosis, and IS6110 as the traditional identification marker for MTBC. The results were compared with those from conventional culture and biochemical identification methods in combination with clinical assessments. Our results showed that the multiplex nested PCR-ICT assay is a convenient, low-cost and easy-to-use detection system for identification of MTB with high sensitivity and specificity. 2. Materials and methods 2.1. Specimen collection and processing A total of 1500 sequential clinical sputum specimens were collected from the mycobacteriology Laboratory, Department of Laboratory Medicine, National Taiwan University Hospital from the September 2004 to March 2005. Collection of these clinical samples was approved by the Review Board Committee in the National Taiwan University Hospital. Specimens were processed on receipt according to the standard routine diagnosis procedures (Piersimoni et al., 2002; Wang et al., 2004). Briefly, an equal volume of NaOH-citrate-N-acetyl-Lcysteine solution was added into sputum sample at room temperature for 15min. After centrifugation, the precipitate was resuspended in 1ml of phosphate-buffered saline (pH 7.4). 2.2. Culture and biochemical methods for diagnosis of MTBC and MTB The LowensteinJensen (LJ) slants (Difco, USA) and Middlebrook 7H11 medium plates (Becton-Dickinson, USA) were inoculated with 250 l of decontaminated sample suspension, incubated at 37C with 5% CO2. An inverted light microscope was used for observation of mycobacterial growth during weeks 2 8 after inoculation. The guidelines of US Center for Disease Control and Prevention (Montenegro et al., 2003) were followed for determination of positive mycobacterial growth. For maximum isolation Mycobacteria growth indicator tubes, the fluorometric BACTEC technique (BACTEC MGIT 960 system; BectonDickinson Diagnosis Instrument System, USA) was used for mycobacterial growth before further growth on 7H11 medium (O'Sullivan et al., 2002). Identification of the bacterial strains to be MTBC and MTB is mainly

442 Table 1 Mycobacterium species tested Reference strains

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2.4. Chromosomal DNA preparation and Multiplex nested PCR


Multiplex nested PCR-ICT IS6110 Rv3618

Mycobacterium spp. M. tuberculosis H37Rv (ATCC 27294) M. bovis (ATCC 19210) M. microti (ATCC 19422) M. avium-intracellulare complex (ATCC 35761) M. kansasii (ATCC 12478) M. marinum (ATCC 927) M. chelonae (ATCC 35752) M. abscessus (ATCC 19977) M. fortuitum (ATCC 6841) M. smegmatis (ATCC 35798) M. xenopis (ATCC 19250) M. asiaticum M. haemophilum M. mucogenicum M. malmoense M. terrae M. triviae M. vaccae M. flavescence M. gastri M. gordonae M. scrofulaceum M. simiae

+ + +

Reference strains were either ATCC strains or clinically isolated strains identified by biochemical assays and 16S rDNA sequencing.

based on the routine morphological and biochemical assays (Parsons et al., 2002). 2.3. Mycobacterium ATCC reference strains A total of 23 Mycobacterium ATCC reference strains (Table 1) were used as controls in the multiplex nested PCR assay.

Decontaminated sample suspensions (100300 l) were mixed with equal volume of wash buffer (TrisHCl buffer, pH = 8). After vortexing for 20 s, samples were subject for centrifugation at 13000 rpm for 10 min before discarding the supernatant. The precipitated pellet was resuspended and lysed in lysis buffer (KOH, pH 13.1) at 95C for 15 min before being neutralized by neutralization buffer (HCl and acetic acid, pH 1.2)(Wang et al., 2004). A 5 l of crude extract suspension was then transferred to an eppendorf tube containing 50 l of amplification reagent (10 mM TrisHCl pH 8.3, 50mM KCl, 1.5mM MgCl2, 0.2 mM dNTP, 20pmol for each primer and 2.5 units Taq DNA polymerase (Takara, Japan)) for multiplex nested PCR. Primers (Table 2) used in nested PCR were designed from IS6110 (Noordhoek et al., 1996; Salo et al., 1994) or Rv3618 belonging to RD9 (Behr et al., 1999) in the M. tuberculosis genome. The multiplex nested PCR was carried out in a thermal reactor (Biometra, Germany). After a 5 min incubation at 94 C, the longer DNA fragments (245 bp for IS6110 and 326 bp for Rv3618) were first amplified by primer pairs INS1INS2 and Rv3618FRv3618R, respectively, under the reaction conditions of 40 cycles at 94C for 30 s, 64C for 15 s, and 72 C for 30 s. Samples were incubated for 3 min at 72C after the last cycle. The PCR mixtures without template DNA were used as the negative controls. Subsequently, 5 l of PCR mixture from first PCR reaction was used for the second PCR reaction (10 mM TrisHCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2mM dNTP, 20pmol for each primer and 2.5 units Taq DNA polymerase (Takara, Japan)). A total of 14 cycles were performed to amplify the 110 bp (for IS6110) and 224 bp (for Rv3618) fragments by using the primer pair B-INS1

Table 2 DNA sequences of primers used for PCR amplification of IS6110 gene, Rv3618 gene and RD9 Target DNA IS6110 Primer sequence (5 3) 1st: INS1: CGTGAGGGCATCGAGGTGGC INS2: GCGTAGGCGTCGGTGACAAA 2nd: B-INS1: Biotin-CTCGTCCAGCGCCGCTTCGG F-INS2: Fluorescein-GCGTCGGTGACAAAGGCCAC 1st: Rv3618F: ATTGCACATCCGCCCC Rv3618R: GGACAAACCCTGCCGC 2nd: B-Rv3618: Biotin-GCTCAACACCCGCCAATC D-Rv3618: Dig-ACATCCGCCCCTACACC RD9 FF: GTGTAGGTCAGCCCCATCC RD9 Int: CAATGTTTGTTGCGCTGC RD9 FR: GCTACCCTCGACCAAGTGTT Product size (bp) 245 110 326 224 306 (region present); 206 (region absent) Parsons et al., 2002 Behr et al., 1999 Reference Montenegro et al., 2003; Reischl et al., 1998

Rv3618

RD 9

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INS1 110 bp

INS2

PCR product loading site

IS6110 B-INS1 B F F-INS2 Nested PCR B F B D Nested PCR B-Rv3618 B D D-Rv3618 Rv3618 Rv3618F 224 bp Rv3618R

S B

S B M

F S M Direction of flow

Test line 2 (anti-Digoxigenin)

Test line 1 (anti-FITC)

Control line (anti-mouse IgG)

antibody F fluorescin(FITC) PCR products

D digoxigenin(Dig) B biotin primer

S M

colloidal gold conjugated with streptavidin colloidal gold conjugated with mouse IgG bacterial DNA

Fig. 1. Schematic diagram of multiplex nested PCR combined with immuno-chromatography (ICT) assay for detection of IS6110 and Rv3618 target DNAs specific for Mycobacterium tuberculosis complex (MTBC) and M. tuberculosis (MTB), respectively.

(biotinylated)-F-INS2 (fluorescein-labeled) and BRv3618 (biotinylated)-D-Rv3618 (Digoxigenin-labeled), respectively. Amplified DNA products were checked, respectively, by ICT and electrophoresis in 2% agarose gel followed by ethidium bromide staining. 2.5. Immuno-chromatography (ICT) strip device and assay The ICT strips were manufactured by Tyson Bioresearch Inc. (Taiwan) and were composed of three regions: a fiberglass surface region on which streptavidin-colloidal gold and mouse IgG (immunoglobulin G) antibodycolloidal gold were impregnated, a nitrocellulose membrane surface region on which anti-Dig, anti-FITC and anti-mouse IgG antibodies were coated, and a thickened filter paper region for suction of buffer solutions. Briefly, colloidal gold particles with an average diameter of 40 nm were prepared as described (Tschopp et al., 1982; Tschopp, 1984). The streptavidin (Sigma) was conjugated with colloidal gold and sprayed on the fiberglass. The nitrocellulose membrane contained three independent results reading lines, two for test lines on which anti-Dig (T2) and anti-FITC (T1) antibody were coated, respec-

tively, and one for control line on which goat-anti-mouse IgG antibody was coated. After multiplex nested PCR, 10 l of the amplified DNA solution was dropped onto the sample pad, followed by addition of 35 l flowing buffer (PBS, pH 7.4). The results were read by presence or absence of the control line and the test line(s) after 10min at room temperature. Presence of a control line indicated a normal flow of the liquid through the strip while absence of the control line indicated invalidation of the assay. 2.6. Quality control For quality control of the chromosomal DNA extraction, a fixed amount (about 103 CFU/ml) of MTB bacteria (ATCC 27294) was spiked into the normal sputum sample followed by routine sputum treatment procedures and chromosomal DNA extraction. For subsequent PCR amplification, positive and negative controls were included in all runs, and part of the decontaminated specimens was frozen to allow for an eventual retest. Precautions to avoid PCR contamination were taken (separate laboratories, laboratory coats, and pipettes, filter-plugged tips, decontamination of benchtops with sodium hypochlorite, etc.). All PCR

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controls were within the stipulated limits. For DNA amplification of samples prepared from clinical specimens, an internal DNA control in which a partial spnI DNA fragment amplified by SI-1 and SI-2 primers was included (Horng et al., 2002), which rules out the possibility of false-negative results due to inhibitors from specimen. 2.7. Clinical assessment of TB patients All medical records, including history, symptoms, signs, radiology, pathology, microbiology results and follow-up observations were carefully reviewed as described from our previous study (Wang et al., 2004). Basically, the culture results and clinical evaluations were served as the gold standard for diagnosis. The multiplex nested PCR-ICT results were evaluated based on these parameters. 2.8. Detection of RD9 by PCR A 50 l reaction mixture containing two flanking primers RD9 FF and RD9 FR (10 mM each), an internal primer RD9-Int (50 mM), KCl (50 mM), TrisHCl (10 mM, pH 8.3), MgCl2 (1.5 mM), each deoxynucleoside triphosphate (200 M each), Taq DNA polymerase (2.5 U), and 5 l of crude cell extract (Section 2.4) were used for PCR. After denaturation at 95 C for 5 min, the reaction mixtures were subject for reactions of 40 cycles at 94 C for 30 s, 65 C for 1min, and 72 C for 1 min. 3. Results 3.1. Principle of multiplex nested PCR-ICT assay Design of this assay is described in Fig. 1. For multiplex nested PCR reactions, two set of primer pairs, each containing an external primer pair amplifying a longer DNA fragment and an internal primer pair amplifying a shorter internal DNA fragment, were designed from the chromosomal DNA sequence of the insertion sequence IS6110 and the Rv3618 open reading frame, respectively. All four external primers were not labeled. Two of the four internal primers were labeled with biotin, and each of the other two was either labeled with fluorescein (for IS6110) or digoxigenin (Dig) (for Rv3618) at the 5-terminus, respectively (Fig. 1). For specifically amplifying Rv3618, the external primer pair Rv3618FRv3618R (Table 2) was first used to amplify a 326 bp DNA fragment, which was subsequently used as a template for amplifying an internal 224 bp fragment (Fig. 2A) using the internal primers B-

Rv3618 (biotinylated) and D-Rv3618 (Dig-labeled) (Table 2). For specifically amplifying the IS6110 sequence, the external primer pair INS1INS2 (Table 2) and internal primer pair B-INS1 (biotinylated) and FINS2 (fluorescein-labeled)(Table 2) were used to amplify a 245 and a 110 bp (Fig. 2A) DNA fragment, respectively. Identification of the labeled amplified DNA products was achieved by the ICT strip. Under the condition of formation of a control line where intensified dark brown color deposits were formed, only amplified DNA fragments doubly labeled with Dig-biotin and/or fluorescein-biotin could lead to formation of test line(s)

(A)
bp

1000

500 400 300 200 100

224 bp 110 bp

(B)

C T2 T1

Fig. 2. Typical results of detection of amplified partial IS6110 and Rv3618 DNA fragments by agarose gel electrophoresis and ICT after multiplex nested PCR. (A) Separation of the amplified 110 bp (IS6110) or 224 bp (Rv3618) DNA fragment by 2% agarose gel electrophoresis followed by ethidium bromide staining. M, DNA size marker. (B) ICT strip assay. After PCR, 5 l of the solution was applied onto the strip. Results were read after 10min incubation at room temperature. N, negative control; C, control line; T1, IS6110 detection line; T2, Rv3618 detection line. For both (A) and (B), chromosomal DNAs of Mycobacterium tuberculosis H37Rv (lane 1), M. bovis (lane 2) and M. avium (lane 3), each at the amount of 1ng was used as PCR templates.

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1103

110-1

110 2

1101

1100

(A)
bp

CFU/ml M N

Table 3 Comparison of MTBC diagnosis results from consecutive 1500 clinical sputum specimens by culture and mutiplex nested PCR-ICT (IS6110) assays Mutiplex nested PCR-ICT (no. of samples) Culturea Positive Positive(114) Negative(1386) Overall (1500) 85 4 89 Negative 29 1382 1411

500 300 200 100 Rv3618 IS6110

a Culture and biochemical diagnosis results were from mycobacteriology laboratory, National Taiwan University Hospital (NTUH). Sensitivity: 95.5%, Specificity: 97.9%; positive predictive value: 74.6%, negative predictive value: 99.7%.

(B)

L
C T1 T2

tuberculosis H37Rv. In comparison, only IS6110 test line was present in detection of M. bovis ATCC19210 and M. microti ATCC 19422 which are MTBC members. For the other non-tuberculous Mycobacterium (NTM) strains, negative reactions were observed from both test lines (Table 1 and Fig. 1). Thus besides MTBC, MTB can further be identified by this assay. 3.3. Screens of clinical isolates A total of 1500 consecutive clinical sputum specimens collected during period September 2004 to March 2005 were subject to routine identification of mycobacteria in National Taiwan University Hospital. In parallel, the spent sediments of specimens were subject for chromosomal DNA extraction and detected by the multiplex nested PCR-ICT assay. The results of detection were summarized in Tables 3 and 4. Among the specimens identified by cultures and biochemical methods, a total of 89 specimens were reported to contain MTBC strains. Compared with the routine identification methods, results from the multiplex nested PCR-ICT assay identified 114 specimens containing MTBC strains, showing a 95.5% sensitivity and 97.8% specificity (Table 3). Comparatively, a total of 29 specimens
Table 4 Comparison of MTB diagnosis results from consecutive 1500 clinical sputum specimens by culture and mutiplex nested PCR-ICT (Rv3618) assays Mutiplex nested PCR-ICT (no. of samples) Culturea Positive Positive(83) Negative(1417) Overall (1500) 80 6 86 Negative 3 1411 1414

Fig. 3. Detection limit of multiplex nested PCR-ICT assay. The detection limit of the assay was evaluated by using a serially diluted M. tuberculosis bacteria ranging from 1 103 CFU/ml to 1 10 1 CFU/ml per reaction as the templates. Amplified DNA products were detected by (A) 2% agarose gel electrophoresis followed by ethidium bromide staining, and (B) the ICT strip assay. M: DNA size marker. N, PCR negative control. L, lateral flow negative control. C, positive control line, T1, Rv3618 detection line, T2, IS6110 detection line.

(Figs. 1 and 2B). The amplified 110bp DNA product interacted with anti-FITC antibodies, forming IS6110 test line (T1), and the amplified 224 bp fragment interacted with anti-Dig antibodies, forming Rv3618 test line (T2) on the strip (Fig. 2B). Formation of both test lines (IS6110 and Rv3618) indicated existence of MTB DNA in the specimen, and formation of IS6110 test line only indicated MTBC. Formation of no test lines indicated either no Mycobacterium bacteria, or alternatively existence of non-MTBC (non-tuberculosis Mycobacterium, NTM) organisms, such as Mycobacterium avium in the specimen (Fig. 2B). The detection limit of this assay system is up to 10 CFU per reaction. (Fig. 3). 3.2. Detection of Mycobacterium reference strains A total of 23 Mycobacterium spp. reference strains grown on 7H11 culture plates were first identified by the multiplex nested PCR-ICT assay. While control lines all showed positive reaction results, both IS6110 and Rv3618 test lines were formed in detection of M.

a Culture and biochemical diagnosis results were from mycobacteriology laboratory, NTUH. Sensitivity: 93%, specificity: 99.8%; positive predictive value: 96.4%, negative predictive value: 99.6%.

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that were MTBC culture-negative were detected as IS6110-positive. Further clinical assessment showed that 3 out of the 29 patients showed significant clinical syndromes of MTB infection. Among the 89 culture positive MTBC specimens, 4 were IS6110-negative based on the multiplex nested PCR-ICT assay. Among the 1500 clinical specimens, 86 samples were MTB positive by culture assay. In comparison, 83 specimens were detected Rv3618 positive by the multiplex nested PCR-ICT assay (Table 4). There were 3 MTB culture-negative culture samples that were detected Rv3618 positive by this assay, and all 3 patients showed significant clinical syndromes of MTB infection. A total of 6 MTB culture-positive specimens were Rv3618 negative by this assay. Briefly, a sensitivity of 93% and specificity of 99.8% were obtained. Among the 85 specimens which were IS6110 positive and culture confirmed to contain MTBC, 80 contained MTB and 5 did not show Rv3618 positive, suggesting that these five specimens contain either MTBC but not MTB or MTB deficient in RD9 (Rv3618) region. 3.4. Absence of RD9 from two MTB strains To further confirm these 5 Rv3618-negative strains were indeed RD9-deficient, three primers designed from RD9 region (Table 2) were used in PCR for confirmation. Among these, RD9 FF and RD9 FR were designed from sequences flanking the RD9 region and RD9-Int from the internal RD9 region (Parsons et al., 2002). Absence of RD9 lead to amplification of a 206 bp DNA

product by primer pair RD9 FF/RD9 FR. Comparatively, a 306bp DNA was amplified by primer pair RD9 FF/ RD9-Int, when RD9 is present (Parsons et al., 2002). No RD9 homologous DNA fragment was detected in these 5 Rv3618-deficient MTBC strains (Fig. 4), which was in agreement with the multiplex nested PCR-ICT assay results. Through culture method, morphology observation and biochemical tests, two bacterial isolates were finally classified as MTB and three as M. bovis. This finding was supported by the results from reference strains identification (Table 1 and Fig. 1) and the study by Parsons et al. (2002). Briefly, 2.3% (2/86) of clinically isolated MTB strains in Taiwan were RD9 absent in this study. 4. Discussion Prompt diagnosis of pulmonary tuberculosis is critical for initiating appropriate therapy and facilitating measures to prevent dissemination of this contagious disease. While MTB is the main devastating pathogen leading to tuberculosis, other members of the MTBC contribute to diseases of different host ranges, geographical prevalences and pathogenesis (Niemann et al., 2004). As more and more MTBC strains are isolated from different region, besides diagnosis of MTBC, it is important to further distinguish MTB from other members in MTBC. However, the currently commonly used molecular diagnosis methods do not achieve such distinction, due to use of the target DNA markers such as IS6110 and 16S rDNA sequences. Comparative genomics of the members of MTBC by use of subtractive hybridization (Mahairas et al., 1996), bacterial artificial chromosome arrays (Brosch et al., 1998; Gordon et al., 1999) or whole genome DNA microarrays (Behr et al., 1999) had identified 16 regions ranging in size from 2 to 12.7kb that were present in M. tuberculosis H37Rv but absent in most BCG derivatives and other members of the MTBC. Among the RD regions analyzed, PCRbased genomic deletion analysis further showed that RD9 seems to be a good DNA marker for specific identification of M. tuberculosis from other members of MTBC (Behr et al., 1999; Parsons et al., 2002). Based on these observations, we chose Rv3618 from the RD9 region as the potential DNA marker for MTB diagnosis. The results obtained basically agreed with what from culture and biochemical assays, suggesting Rv3618 is a good marker for MTB. Among the 85 T1-positive MTBC specimens, 80 were also T2-positive. Three of these 5 specimens were subsequently identified to contain M. bovis and the remaining 2 to be MTB by culture and biochemical assays. Further confirmation of

M 1
bp

1000 500 400 300 200 100

306 bp 206 bp

Fig. 4. Detection of RD9 by PCR. After PCR using chromosomal DNA as the template and primers listed in the Table 2, DNAs were separated in 2% agarose gel electrophoresis followed by ethidium bromide staining. Observation of a 306 bp PCR product indicates presence of RD9 and a 206 bp product indicates deletion of this region. M, DNA size marker; lane 12, clinical MTB isolates (IS6110positive/Rv3618-negative); lane 35, clinical M. bovis isolates (IS6110-positive/Rv3618-negative); lane 6, M. tuberculosis H37Rv and lane 7, M. bovis (ATCC 19210).

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absence of RD9 within these 2 MTB strains was achieved by a PCR protocol described by Parsons et al. (2002). These data indicated that while RD9 may not exist in other MTBC species, a part of MTB strains isolated in Taiwan did not contain RD9. This should be taken into consideration when using Rv3618 as a marker to detect MTB. Although a range of rapid tests based on nucleic acid amplification techniques have been developed for direct detection of MTBC from clinical samples, it remains to be proven whether they can also fulfill the requirements of high sensitivity and specificity, simplicity and reasonable cost at the same time. The relative high cost of most molecular methods developed maybe one of the major reasons that hinder these systems from being widely used, especially in developing countries where TB is much more prevalent than the developed ones. The multiplex nested PCT-ICT assay developed in this laboratory is efficient due to characteristics of easy to operate, no need of sophisticated detection equipment, low cost, time saving (less than 4 h from receipt of sputum specimens to completion of the test), and high sensitivity and specificity. Compared with conventional culture and biochemical diagnosis methods, the sensitivity and specificity of this assay for MTBC diagnosis are 95.5% and 97.9%, respectively, and for MTB are 93% and 99.8%, respectively. The results are comparable to, or even better than those obtained by previous systems we used for MTBC diagnosis (Wang et al., 2004). Among the 89 culture identified MTBC specimens, 85 were IS6110-positive. There were 4 specimens that are neither MTBC nor MTB positive by this assay. These might be due to too few bacterial cells within the sputum specimens. There were also 5 specimens which were IS6110-positive while Rv3618-negative. Among these, 3 were finally confirmed to contain M. bovis and 2 MTB without RD9. On the contrary, there were 29 culture-negative but IS6110-positive samples, and 3 culture-negative but IS6110 and RV3618-positive samples. For the 3 culture-negative but IS6110 and Rv3618 positive samples, retrospective clinical assessment indicated that the 3 patients were infected by MTB before and had been under drug treatment when samples were taken. Thus the results might be due to killing of MTB cells by drug or full recovery. The remaining 26 culturenegative but IS6110-positive samples might be due to non-specifically amplified DNA products during the PCR-ICT assay. Although the multiplex nested PCR and ICT techniques have already been widely used in the areas of molecular and immuno-diagnosis, few reports are on combination of both techniques into an assay for rapid

diagnosis of specific DNA target sequences. A recent report from Takada et al. (2005) shows a great potentiality of such technique on direct diagnosis of Porphyromonas gingivalis from periodontitis patients (Takada et al., 2005). Some other techniques which also detect target nucleic acid sequence by lateral-flow device (strip) are also developed. These include cycling probe technology (CPT) assay with a lateral-flow device for detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures (Fong et al., 2000). Another example is the up-converting phosphor reporters and lateral-flow assay to identify human papillomavirus type16 (Corstjens et al., 2001). Comparatively, our system has the advantage of direct and simultaneous detection of two DNA targets, which is convenient even for use in identification of different pathogen in one sample. Low cost, simple accurate, the potential of high through detection of target DNA sequences are the main characteristics of these systems. Furthermore, direct detection from clinical samples with comparable sensitivity and specificity with conventional or commercial systems are also highlighted. These are especially important for practical clinical applications. Acknowledgments This work was supported by the grant from Tyson bioresearch Inc. (SPA Examination Committee R and D Project, Grand No. 692 ) and the Technology Development Program for Academia, Ministry of Economical Affairs (grant number 91-EC-17-A-10-S1-0013), which were really appreciated. References
Abe, C., Hirano, K., Wada, M., Kazumi, Y., Takahashi, M., Fukasawa, Y., Yoshimura, T., Miyagi, C., Goto, S., 1993. Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J. Clin. Microbiol. 31, 32703274. Alcaide, F., Benitez, M.A., Escriba, J.M., Martin, R., 2000. Evaluation of the BACTEC MGIT 960 and the MB/BacT systems for recovery of mycobacteria from clinical specimens and for species identification by DNA AccuProbe. J. Clin. Microbiol. 38, 398401. Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S., Small, P.M., 1999. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 15201523. Boddinghaus, B., Rogall, T., Flohr, T., Blocker, H., Bottger, E.C., 1990. Detection and identification of mycobacteria by amplification of rRNA. J. Clin. Microbiol. 28, 17511759. Brosch, R., Gordon, S.V., Billault, A., Garnier, T., Eiglmeier, K., Soravito, C., Barrell, B.G., Cole, S.T., 1998. Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect. Immun. 66, 22212229.

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P.-C. Soo et al. / Journal of Microbiological Methods 66 (2006) 440448 tuberculosis: an international collaborative quality control study among 30 laboratories. J. Clin. Microbiol. 34, 25222525. O'Sullivan, C.E., Miller, D.R., Schneider, P.S., Roberts, G.D., 2002. Evaluation of Gen-Probe amplified mycobacterium tuberculosis direct test by using respiratory and nonrespiratory specimens in a tertiary care center laboratory. J. Clin. Microbiol. 40, 17231727. Parsons, L.M., Brosch, R., Cole, S.T., Somoskovi, A., Loder, A., Bretzel, G., Van Soolingen, D., Hale, Y.M., Salfinger, M., 2002. Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis. J. Clin. Microbiol. 40, 23392345. Piersimoni, C., Callegaro, A., Scarparo, C., Penati, V., Nista, D., Bornigia, S., Lacchini, C., Scagnelli, M., Santini, G., De Sio, G., 1998. Comparative evaluation of the new gen-probe Mycobacterium tuberculosis amplified direct test and the semiautomated abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens. J. Clin. Microbiol. 36, 36013604. Piersimoni, C., Scarparo, C., Piccoli, P., Rigon, A., Ruggiero, G., Nista, D., Bornigia, S., 2002. Performance assessment of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex from respiratory and extrapulmonary specimens. J. Clin. Microbiol. 40, 41384142. Reischl, U., Lehn, N., Wolf, H., Naumann, L., 1998. Clinical evaluation of the automated COBAS AMPLICOR MTB assay for testing respiratory and nonrespiratory specimens. J. Clin. Microbiol. 36, 28532860. Salo, W.L., Aufderheide, A.C., Buikstra, J., Holcomb, T.A., 1994. Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy. Proc. Natl. Acad. Sci. U. S. A. 91, 20912094. Soini, H., Musser, J.M., 2001. Molecular diagnosis of mycobacteria. Clin. Chem. 47, 809814. Sreevatsan, S., Pan, X., Stockbauer, K.E., Connell, N.D., Kreiswirth, B.N., Whittam, T.S., Musser, J.M., 1997. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. Natl. Acad. Sci. U. S. A. 94, 98699874. Takada, K., Sakaguchi, Y., Oka, C., Hirasawa, M., 2005. New rapid polymerase chain reaction-immunochromatographic assay for Porphyromonas gingivalis. J. Periodontol. 76, 508512. Tschopp, J., 1984. Ultrastructure of the membrane attack complex of complement. Heterogeneity of the complex caused by different degree of C9 polymerization. J. Biol. Chem. 259, 78577863. Tschopp, J., Podack, E.R., Muller-Eberhard, H.J., 1982. Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8. Proc. Natl. Acad. Sci. U. S. A. 79, 74747478. Wang, J.Y., Lee, L.N., Chou, C.S., Huang, C.Y., Wang, S.K., Lai, H.C., Hsueh, P.R., Luh, K.T., 2004. Performance assessment of a nestedPCR assay (the RAPID BAP-MTB) and the BD ProbeTec ET system for detection of Mycobacterium tuberculosis in clinical specimens. J. Clin. Microbiol. 42, 45994603. Woods, G.L., 1999. Molecular methods in the detection and identification of mycobacterial infections. Arch. Pathol. Lab Med. 123, 10021006. Yuen, K.Y., Yam, W.C., Wong, L.P., Seto, W.H., 1997. Comparison of two automated DNA amplification systems with a manual onetube nested PCR assay for diagnosis of pulmonary tuberculosis. J. Clin. Microbiol. 35, 13851389.

Brosch, R., Gordon, S.V., Marmiesse, M., Brodin, P., Buchrieser, C., Eiglmeier, K., Garnier, T., Gutierrez, C., Hewinson, G., Kremer, K., Parsons, L.M., Pym, A.S., Samper, S., van Soolingen, D., Cole, S.T., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. U. S. A. 99, 36843689. Brown, T.J., Power, E.G., French, G.L., 1999. Evaluation of three commercial detection systems for Mycobacterium tuberculosis where clinical diagnosis is difficult. J. Clin. Pathol. 52, 193197. Caws, M., Wilson, S.M., Clough, C., Drobniewski, F., 2000. Role of IS6110-targeted PCR, culture, biochemical, clinical, and immunological criteria for diagnosis of tuberculous meningitis. J. Clin. Microbiol. 38, 31503155. Corstjens, P., Zuiderwijk, M., Brink, A., Li, S., Feindt, H., Niedbala, R.S., Tanke, H., 2001. Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papillomavirus type 16 infection. Clin. Chem. 47, 18851893. Eing, B.R., Becker, A., Sohns, A., Ringelmann, R., 1998. Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis assay with in-house PCR and culture for detection of M. tuberculosis. J. Clin. Microbiol. 36, 20232029. Fong, W.K., Modrusan, Z., McNevin, J.P., Marostenmaki, J., Zin, B., Bekkaoui, F., 2000. Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology. J. Clin. Microbiol. 38, 25252529. Gardiner, D.F., Beavis, K.G., 2000. Laboratory diagnosis of mycobacterial infections. Semin. Respir. Infect. 15, 132143. Gordon, S.V., Brosch, R., Billault, A., Garnier, T., Eiglmeier, K., Cole, S.T., 1999. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol. Microbiol. 32, 643655. Hellyer, T.J., Fletcher, T.W., Bates, J.H., Stead, W.W., Templeton, G.L., Cave, M.D., Eisenach, K.D., 1996. Strand displacement amplification and the polymerase chain reaction for monitoring response to treatment in patients with pulmonary tuberculosis. J. Infect. Dis. 173, 934941. Horng, Y.T., Deng, S.C., Daykin, M., Soo, P.C., Wei, J.R., Luh, K.T., Ho, S.W., Swift, S., Lai, H.C., Williams, P., 2002. The LuxR family protein SpnR functions as a negative regulator of Nacylhomoserine lactone-dependent quorum sensing in Serratia marcescens. Mol. Microbiol. 45, 16551671. Katila, M.L., Katila, P., Erkinjuntti-Pekkanen, R., 2000. Accelerated detection and identification of mycobacteria with MGIT 960 and COBAS AMPLICOR systems. J. Clin. Microbiol. 38, 960964. Mahairas, G.G., Sabo, P.J., Hickey, M.J., Singh, D.C., Stover, C.K., 1996. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178, 12741282. Montenegro, S.H., Gilman, R.H., Sheen, P., Cama, R., Caviedes, L., Hopper, T., Chambers, R., Oberhelman, R.A., 2003. Improved detection of Mycobacterium tuberculosis in Peruvian children by use of a heminested IS6110 polymerase chain reaction assay. Clin. Infect. Dis. 36, 1623. Niemann, S., Kubica, T., Bange, F.C., Adjei, O., Browne, E.N., Chinbuah, M.A., Diel, R., Gyapong, J., Horstmann, R.D., Joloba, M.L., Meyer, C.G., Mugerwa, R.D., Okwera, A., Osei, I., OwusuDarbo, E., Schwander, S.K., Rusch-Gerdes, S., 2004. The species Mycobacterium africanum in the light of new molecular markers. J. Clin. Microbiol. 42, 39583962. Noordhoek, G.T., van Embden, J.D., Kolk, A.H., 1996. Reliability of nucleic acid amplification for detection of Mycobacterium

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