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As presented at the 99th General Meeting of the American Society for Microbiology, May 1999. The Development of Three Strand Displacement Amplification Assays for Culture Confirmation of Mycobacterium tuberculosis Complex, Mycobacterium avium Complex and Mycobacterium kansasii on the BDProbeTec ET* System TM G. DURMOWICZ, T. BRINK JR, S. BUSTOS, D. COPERTINO, C. DULANY, J.M. HARRIS, K. KELLY, O. LLORIN, J. PRICE, AND K. YANSON BD Biosciences • 54 Loveton Circle • Sparks, MD, USA 21152 REVISED ABSTRACT s The clinical importance of mycobacterial infections is increasing, particularly as a result of AIDS. The diagnosis of these infections has been traditionally dependent on culture followed by biochemical analysis. These procedures are time consuming and labor intensive; diagnosis can take as long as six weeks. In combination with automated culturing systems such as the BACTECTM460TB and the BACTEC MGITTM 960 Systems, a nucleic acid amplification test can significantly reduce the time to diagnosis. Here we report the development of three homogeneous strand displacement amplification (SDA) assays for the detection of either Mycobacterium tuberculosis complex (Mtb), Mycobacterium avium complex (MAC) or Mycobacterium kansasii from culture. Each assay simultaneously amplifies and detects specific target DNA and an internal amplification control. Assay performance was optimized by evaluating different reaction components of the systems in statistically designed experiments. These experiments identified a common sample buffer, thereby allowing one sample to be tested in each of the three assays. INTRODUCTION Sensitivities of the assays were evaluated across multiple Mycobacterium tuberculosis (Mtb) and nontuberculous strains of organisms. The Mtb, MAC and M. kansasii assays (NTM) mycobacterial diseases pose an increasing public health challenge. Mtb and NTM infections frequently occur in patients demonstrated sensitivities of 100% (10/10), 100% (50/50) with AIDS or other immunocompromised illnesses. Mycobacterium and 100 (137/137), respectively. An analytical sensitivity avium complex (MAC) and M. kansasii are the two leading causes of NTM infections in humans. The diagnosis of Mtb and NTM using plasmid target DNA was estimated for each assay: infections has been traditionally dependent on acid fast staining M. tuberculosis = 54 copies/reaction, M. avium = 771 copies/ and culture of organisms followed by biochemical analysis. These reaction, M. intracellulare = 58 copies/reaction and methods can be either insensitive or require weeks to perform. Even with automated culturing systems such as the BACTECTM M. kansasii = 155 copies/reaction. These sensitivities allow for 460TB and BACTECTM MGITTM 960 Systems (BD Biosciences, the identification of these organisms on the BACTECTM 460TB Sparks MD), time to identification usually extends to greater than two weeks. Amplification technologies have allowed for the and BACTEC MGITTM 960 Systems at a Growth Index of ≥ 50 increase in sensitivity and specificity as well as decreased time to and Growth Units of ≥ 75, respectively. The specificity of each organism identification. assay was evaluated against 56 other mycobacterial and Here we report the development of three assays for the detection of M. tuberculosis complex, Mycobacterium avium non-mycobacterial species at 108 genome equivalents/ml. complex or M. kansasii on the BDProbeTecTM ET System. The No significant crossreactivity to any clinically relevant assays utilize homogeneous strand displacement amplification (SDA) to simultaneous amplify and detect a target region of the mycobacterial or non-mycobacterial species tested was IS6110, dna J, or KATS1 region of the genome for the observed in any of the three assays. The culture confirmation identification of Mtb, MAC, and M. kansasii organisms, assays on the BDProbeTecTM ET System are sensitive, specific respectively. The assays also include an internal amplification control (IAC) to validate negative results. Optimization of the and can be used for the identification of Mtb, MAC and M. assays has allowed for the testing of all three organisms from a kansasii from a single culture specimen in less than four hours. single culture aliquot with time to results in under four hours. *Product under development METHODS Figure 1. Mechanism of SDA — Target Generation Step SDA Mechanism SDA amplification can be broken down into two phases. The first phase, 1. Mtb, MAC or M. kansasii DNA the Target Generation step (Figure 1), creates the structure that feeds into the denature target DNA and second phase, the Exponential Amplification step (Figure 2). Target DNA is bind primers denatured during the lysis step. Before reannealing can occur, the sample is 2. 4. mixed with an excess of two primers (S1 and S2), two bumpers (B1 and B2), and a detector probe (Figure 1, steps 1 and 2). Primers consist of a target specific DNA hybridizing region at the 3' end, a non-hybridizing tail at the 5' 3. end, and a recognition site for the restriction enzyme BsoBI (CTC GGG, indicated by in Figures 1 & 2) between these two regions. Bst 5. polymerase and restriction enzyme are added after the priming step. Both primers and bumpers are extended by the polymerase (Figure 1, step 3). In the process of extending the bumpers, the polymerase displaces the primer extension product. This extension product next hybridizes to a complementary primer and bumper (Figure 1, step 4), and extension and Figure 2 displacement follows. The resulting strands can be hybridized to complementary primers, and these primers can be extended. The end products (Figure 1, step 5) contain the detector probe annealing region Figure 2. Homogeneous SDA Detection flanked by nickable BsoBI sites. The primers contain the restriction sequence CTC GGG. During extension, the polymerase incorporates alpha thio-dCTP to create the 1. complement of the restriction site. The result is a hemiphosphorothioated restriction site, which can only be nicked, not cut completely. Therefore, the 2. products of step 5 can be nicked by BsoBI. The polymerase will extend from the nicked site, displacing the fragments designated as T1 and T2, which will 3. feed in to the second phase, Exponential Amplification (Figure 2, left side). Detection 4. Detection occurs simultaneously with amplification. The detector probe consists of a target specific hybridization region at the 3' end, and a hairpin 5. structure at the 5' end. The loop of the hairpin contains the BsoBI recognition sequence CCC GAG. The 5' base is conjugated to donor 6. molecule, while the 3' base of the hairpin stem is conjugated to an acceptor Target or Internal Control Detection molecule. In its native state, the hairpin maintains the donor and the acceptor molecules in close proximity. When the donor is excited, the fluorescent energy is transferred to the acceptor molecule, and little fluorescence is observed. As the hairpin anneals to the target, it is extended by the polymerase, and is displaced by the extension of an upstream primer Figure 3. A simple easy to follow workflow. (Figure 2, step 1-3). The resulting detector extension product is Sample Processing Workflow complementary to the downstream primer S2 (step 4). The primer (S2) is extended (step 5) and the hairpin is linearized. This creates a double stranded 500µl of Liquid Media or 1µl loop Solid Media* cleavable restriction site, which is promptly cleaved by BsoBI. This process frees the donor from the quenching effects of the acceptor, and allows 1mL Sample Wash Buffer. vortex fluorescence to be observed. spin for 3 min. decant. NOTE: The detection method for the internal amplification control (IAC) Heat 105° for 30 min. quickspin. is identical to that of the target specific detection, except on the IAC detector 100µl Sample Lysis Buffer. vortex you have a different pair of donor and acceptor molecules so that target and IAC can be differentiated.2 Sonic Bath for 45 min. at 65°C. quickspin. System Optimization 600µl Sample Neutralization Buffer. vortex. quickspin. Optimization of the Mtb, MAC and M. kansasii assays examined multiple components of the SDA reaction. These components include primers, 100µl BDProbeTecTM ET detectors, bicine, potassium, DMSO, glycerol, magnesium and enzymes. *1µl loop in 1ml sample wash buffer, take 10µl and proceed Initial experiments were designed to select the best primer set and detector. Subsequent experiments were designed to focus on optimization of buffer and enzyme conditions resulting in a common sample buffer that could be used in all three assays. Priming wells containing primers, bumpers, Acknowledgements fluorescent detector and IAC and other SDA components are rehydrated Special thanks to Daryl Shank and Dave Wolfe for supplying the with lysed target in sample diluent. Wells are incubated at room temperature DNAs, cell lysates and organisms used for the specificity and crossre- activity evaluations, Bernie Dellone for reagent support. Also to Max for 20 minutes, then transferred to a 70°C heat block. Amplification wells Kuhn and Paula Johnson for their statistical support. containing the restriction enzyme and polymerase, nucleotides and remaining Strains designated with T were generously provided to BDB by Dr. SDA components are pre-warmed for ten minutes at 52°C as the priming Enrico Tortoli, Laboratorio di Microbiologia e Virologia, Ospedale di wells are heated. After ten minutes, the samples are transferred from the Careggi, 50139 Florence, Italy. priming wells to the amplification wells, sealed and then placed in the References BDProbeTec TM ET instrument for one hour. The thermally controlled 1. Centers for Disease Control and Prevention — http: //www.cdc.gov fluorescent reader within the instrument monitors each reaction for the 2. J. G. Nadeau et al., “Detection of Nucleic Acids by Fluorescence generation of amplified products. Quenching”, US Patent 5846726, 12/98 3. Walker, G.T., J.G. Nadeau, C.P. Linn, R.F. Devlin, and W.B. Dandliker. 1996. Strand displacement amplification (SDA) and transient-state fluorescence polarization detection of Mycobacterium tuberculosis DNA. Clinical Chemistry 42:1, 9-13. Table 1. Specificity of M. tuberculosis Complex M. tuberculosis H37Rv M. tuberculosis VA44 SPECIFICITY M. tuberculosis 19 s 10 Mtb complex, 50 MAC and 137 M. kansasii cell lysates were M. tuberculosis 13 tested at 105 genomes per reaction (Tables 1-3). M. tuberculosis 15 s The specificity of the three assays was tested with DNA or cell lysates from potential crossreactants at 1 x 107 genomes per reaction (Table 4). M. africanum ATCC 35711 M. bovis CDC 52 SENSITIVITY M. bovis ATCC 19210 s Plasmid DNA levels were titrated down to determine the sensitivities of each assay. Limit of detection (LOD) was determined (Figure 4). M. bovis BCG CDC 4 s BACTECTM 12B media and BACTECTM MGITTM 960 tubes were inoculated M. microti LCDC 203 with clinical isolates and frozen NALC pellets. Cultures were harvested and • Specificity of the M. tuberculosis processed from the BACTECTM 12B and BACTECTM MGITTM 960 instruments complex assay. Ten strains of the at a GI~50 and a GU~75, respectively (Table 5). M. tuberculosis complex were tested at 105 copies per reaction. All organisms were detected. Table 2. Specificity of MAC Table 3. Specificity of M. kansasii M. avium M. intracellulare DHMH 10435 LCDC 725 LCDC 711 T1493 T3993 T693 11907-300 P-39 ATCC 15987 DHMH 7349 CDC 7 LCDC 712 T1593 T4093 T6993 14816-1424 157 Manten TMC 1419 DHMH (?) CDC 38 T10392 T15993 T4193 T7093 DHMH 1381 CDC 73 T10495 T1689 T4293 T7193 2993 1195 CDC TMC 1473 DHMH 1862 CDC 94 T10592 T1693 T4392 T7793 TMC 1461 W 552* Edgar Boone DHMH 2265 CDC 8 T10792 T17593 T4393 T785 4443-1237 23393 Yandle DHMH 2268 LCDC 713 T1085 T1793 T4492 T793 34540 Darden* Hillberry 1244-9 DHMH2833 LCDC 714 T10892 T18494 T4493 T7993 B-92 4990 O’Connor 12645 DHMH 2839 DHMH NMH-7 T1093 T186 T4693 T8394 6450-204 ATCC 23435 P-54 DHMH 2840 LCDC 715 T10992 T1893 T4791 T8494 DHMH 2843 LCDC 716 T11092 T190 T4793 T8594 14141-1395 6845 Simpson DHMH 9500 LCDC 717 T11192 T1993 T485 T8694 25546-759 AT 545 Findley Leonard-158 DHMH NJ-787 LCDC 719 T11292 T2193 T486 T8794* 6195 TMC 1466 Anderson DHMH 6948-91 LCDC 720 T11593 T2393 T493 T8894 1784-286 ATCC 25122 P-42 DHMH 6084-91 LCDC 721 T11792 T2494 T5 T8994* SJB #2 Wood Duck TMC 1406 JHH OC-7867* LCDC 722 T1185 T2692 T5295 T9094* JHH 1C-3356 LCDC 701 T12192 T2789 T593 T9095 TMc 1462 72-888 13950 JHH 5C-8246 LCDC 702 T12795 T285 T5993 T9194 128 Germany CDC 1217 JHH 1U-4077 LCDC 703 T13293 T3192 T6093 T9294 1602-1965 JHH 1B-249 LCDC 704 T1391 T3391 T6195 T9494 mTMC 1463 DHMH 403 LCDC 707 T1393 T3690 T6693 T933 • Specificity of the MAC SDA assay. 6194 Fifty MAC strains were tested at LCDC 723 LCDC 708 T14592 T3893 T6793 T994 16741 Cardiff 105 copies/reaction except * were LCDC 724 LCDC 709 T1492 T393 T686 13528-1079 tested at 100ng DNA/reaction. • Specificity of the M. kansasii assay. 137 M. kansasii strains were tested at 105 copies/reaction All 50 MAC strains were detected. except * were tested at 100ng DNA/reaction. All 137 M. kansasii strains were detected. 25291 Table 4. Evaluation of Crossreactivity Mycobacteria species Mycobacteria species Non-mycobacteria species M. aichiense ATCC 27280 M. komossense ATCC 33013 Actinomycese israelii ATCC 10049 M. aurum ATCC 23366 M. malmoense ATCC 29571 Actinoplanes auranticolor ATCC 15330 M. bovis CDC 52 M. marinum BD 2324 Corynebacterium diphtheriae ATCC 11913 M. bovis ATCC 19210 M. microti LCDC 203 Corynebacterium pseudodiptheriticum ATCC 10700 M. bovis BCG CDC 4 M. neoaurum ATCC 25795 Corynebacterium xerosis ATCC 373 M. celatum ATCC 51131 M. obuense ATCC 27023 Eubacterium lentum ATCC 43055 M. chelonae TMC 1543 M. paratuberculosis ATCC 19698 Nocardia asteroides ATCC 3308 M. chitae ATCC 19627 M. scrofulaceum BDDIS 2404 Nocardia brasiliensis ATCC 19296 M. chlorophenolicum ATCC 49826 M. scrofulaceum ATCC 19981 Nocardia orieintalis ATCC 19795 M. confluentis ATCC 49920 M. simiae ATCC 15080 Propionibacterium acnes ATCC 6919 M. fortuitum TMC 2808 M. simiae ATCC 25273 Rhodococcus equi ATCC 6939 M. gadium ATCC 27726 M. simiae ATCC 25275 Rhodococcus rhodochrous ATCC 13808 M. gastri ATCC 15754 M. smegmatis ATCC 19420 Streptomyces albus ATCC 3004 M. gilvum ATCC 43909 M. sphagni ATCC 33027 Streptomyces gedanensis ATCC 4880 M. gordonae ATCC 14470 M. szulgai ATCC 23069 Streptomyces griseus ATCC 10137 M. haemophilum ATCC 43160* M. szulgai ATCC 29716 Streptomyces somaliensis ATCC 33201 M. interjectum ATCC 51457* M. tuberculosis VA 44 Streptosporangium viridialbum ATCC 33328 M. intermedium ATCC 51848 M. xenopi ATCC 19250 Streptoverticillium alboverticillatum ATCC 29818 M. kansasii TMC 1201 • No crossreactivity is seen at 108 copies per ml in the Mtb or M. kansasii assays. *Weak crossreactivity is seen in the MAC assay. However, M. haemophilum requires 30-32°C and iron for optimal growth; M. interjectum is rare and concensus on its classification is unresolved. A titration of M. interjectum exhibited negative results at or below 105 copies per mL. Figure 4. LOD Analysis of Mtb, MAC and M. kansasii Tb Avium 1.0 1.0 0.8 0.8 Proportion Positive Proportion Positive 0.6 0.6 LOD: 54 copies/rxn LOD: 771 copies/rxn 0.4 0.4 0.2 0.2 0.0 0.0 0 30 55 80 105 130 155 0 500 1000 1500 2000 copies/rxn copies/rxn Intracellulare Kansasii 1.0 1.0 0.8 0.8 Proportion Positive 0.6 Proportion Positive 0.6 LOD: 58 copies/rxn LOD: 155 copies/rxn 0.4 0.4 0.2 0.2 0.0 0.0 0 50 100 150 200 250 300 0 200 400 600 800 1000 copies/rxn copies/rxn The LOD is defined as the number of targets that are amplified and detected with 95% probability. CONCLUSION Table 5. BDProbeTecTM ET System Culture Identification from Liquid and Solid Media s Three homogeneous SDA assays with internal amplification controls have been developed for the BACTEC 12B BBL MGIT 960 L-J & 7H11 Solid detection of M. tuberculosis, MAC and M. kansasii GI Range Detected GU Range Detected Detected from a single culture specimen. Color coded Mtb complex 38-360 18/18 53-535 13/13 36/36 microwells containing dried reagents, sealed reaction MAC 12-101 16/16 68-132 16/16 46/46 vessels and rapid time to results makes these assays M. kansasii 12-360 11/11 46-431 8/8 20/20 and the BDProbeTecTM ET System a versatile, user friendly platform. • The system detected 184/184 samples from BACTEC 12B, BBL MGIT 960, Lowenstein-Jensen (L-J) and Middlebrook 7H11 agar cultures. s The three assays demonstrate excellent sensitivity and specificity. All 82/82 (100%) of broth positive cultures from BACTECTM 12B or BACTECTM MGITTM 960 tested and all 102/102 (100%) of L-J and 7H11 solid media have been identified correctly. No crossreactivity was seen in the Mtb or M. kansasii assays. Weak crossreactivity was seen in the MAC assay with two non-clinically relavent mycobacteria species. s A single buffer has been optimized for all three assays, with a sample processing workflow that is simple, easy to follow and compatible with a variety of solid and liquid culture media. In addition samples are rendered non-viable early in the processing to ensure safety and flexibility for the technician. Reprint LR602 Please refer to other posters at this meeting on the performance of the other assays on the BDProbeTecTM ET System.
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