JCM Accepts, published online ahead of print on 8 August 2007 J. Clin. Microbiol. doi:10.1128/JCM.02289-06 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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Evaluation the Invader Assay with the BACTEC MGIT 960 System for Prompt Isolation
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and Identification of Mycobacteria from Clinical Specimens
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Sadahiro Ichimura1, 3, Makoto Nagano2, Nobuko Ito2, Masahiro Shimojima1, Toru Egashira2,
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Chikara Miyamoto2, Kiyofumi Ohkusu3, and Takayuki Ezaki3
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Department of Microbiology, BML, Inc., 2Division of Advanced Technology, BML, Inc., 1361–1
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Matoba, Kawagoe, Saitama 350–1101, Japan;
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Graduate School of Medicine, Gifu, Gifu 501–1194, Japan
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Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University
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*Corresponding author. Mailing address: Department of Microbiology, BML, Inc., 1361–1
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Matoba,
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+81–049–232–0529. E–mail: ichi–
[email protected].
Kawagoe,
Saitama
350–1101,
Japan.
Phone:
+81–049–232–0940.
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Running title: IDENTIFYING MYCOBACTERIA BY INVADER ASSAY
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Key words: mycobacteria, MGIT, 16S rDNA, ITS-1 region, Invader assay 1
Fax:
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ABSTRACT
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Rapid and accurate identification of mycobacterial species is essential for patient management.
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This study describes the use of the Invader assay in conjunction with the BACTEC MGIT 960
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system that provides an efficient procedure in clinical use. This assay discriminates single–base
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differences (e.g., genotyping single nucleotide polymorphisms) under homogeneous and
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isothermal conditions, and can measure directly on genomic DNA without prior target DNA
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amplification. To identify a wide variety of mycobacterial species, 20 Invader probes were
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designed to target the 16S rRNA gene (16S rDNA) and the 16S–23S rRNA gene internal
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transcribed spacer 1 (ITS-1) region. To validate the Invader probes, we used 78 ATCC strains,
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and 607 clinical mycobacterial strains, which were identified by DNA sequencing of the 16S
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rDNA and ITS-1. The Invader assay could accurately identify and differentiate these strains
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according to target sequences. Besides, it could detect and identify 116 (95.1%) of 122 positive
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liquid cultures from the BACTEC MGIT 960 system, and did not react to 83 contaminated
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MGIT cultures. Species identification takes 6.5 h with the Invader assay: 2.0 h for DNA
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extraction, 0.5 h for handling, and up to 4 h for the Invader reaction. The Invader assay has the
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speed, ease of use, and the accuracy to be an effectively procedure for bacteriological diagnosis
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of mycobacterial infections.
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INTRODUCTION
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Rapid and accurate identification of mycobacteria is essential for determining appropriate
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therapies and for epidemiological studies (7, 11, 14, 37). For example, the U.S. Centers for
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Disease Control and Prevention (CDC) recommends turnaround times of 2–3 weeks for
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processing of Mycobacterium tuberculosis (4, 33, 36). A definitive diagnosis of mycobacterial
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infection depends on growth and identification of the bacteria (2, 3). To speed the bacterial
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culturing, time-consuming cultures on egg-based solid media, such as Löwenstein–Jensen (LJ)
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and Ogawa slants, are being replaced by faster liquid culture methods, such as the BACTEC
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MGIT 960 system (Becton Dickinson, Sparks, MD) and the MB/BacT system (Organon Teknika,
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Boxtel, The Netherlands) (1, 13, 15, 21, 22, 33).
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Now a method is urgently needed to rapidly identify a wide variety of Mycobacterium species
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directly from liquid cultures. Unfortunately, the available methods have several limitations. For
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example, the widely used AccuProbe system (GenProbe, San Diego, CA) (8, 21, 25, 29)
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identifies only a limited number of the many mycobacterial species seen in a clinical laboratory.
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While much faster and more accurate, DNA sequencing (16, 26, 39), PCR restriction fragment
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length–polymorphism (RFLP) assays (6, 28, 35) and InnoLiPA Mycobacteria (Innogenetics,
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Ghent, Belgium) (23, 38) require expensive equipment and an exclusive workspace for PCR. In
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addition, DNA sequencing and PCR–RFLP require pure cultures, and if the sample was the
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mixed culture, separate culture on a solid medium would further slow these assays.
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Many routine procedures in clinical laboratories have been simplified by homogeneous
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fluorescent detection systems. Here we report our study of one of these, the Invader assay (Third 3
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Wave Technologies, Madison, WI) (17). The Invader assay can accurately discriminate
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single–base differences, such as single nucleotide polymorphisms (SNPs), and can measure
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directly on genomic DNA without prior target amplification. We evaluated the suitability of the
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Invader assay for directly identifying mycobacteria from the MGIT 960 system.
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MATERIALS AND METHODS
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Bacterial strains. The validation of the Invader probes was performed by testing 78 ATCC
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strains (Table 2), and 607 clinical mycobacterial strains (Table 3), which were identified and
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confirmed the target sequences by DNA sequencing of the 16S rDNA and ITS-1. These clinical
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strains cultured on Ogawa slants mainly for NTM (nontuberculous mycobacterium) infections
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were tested in the BML General Laboratory from December 2003 to June 2005 (excluding
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overlapping patients).
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Clinical specimens for the BACTEC MGIT 960 system. Between November 21 and
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November 25, 2004, 1390 consecutive clinical specimens were received for routine
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mycobacterial detection in the BML General Laboratory. These included 1040 sputum and 155
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other respiratory specimens, 28 digestive samples, 15 sterile body fluids, 14 urine samples, 6
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wound samples, and 132 samples from unspecified sources.
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Inoculation and cultivation of clinical specimens. Before inoculation, the MGIT 960 tubes
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were prepared as described by the manufacturer (Becton Dickinson). A 0.5 ml portion of the
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processed specimen was inoculated into the MGIT, and the tubes were introduced into the MGIT
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960 instrument and incubated until they were found to be positive by the instrument or for 42
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days. A 0.1-ml portion of the processed specimen was inoculated onto a solid slant of Ogawa egg
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medium (Kyokuto Pharmaceutical, Tokyo, Japan), and the slants were incubated in 5–10% CO2.
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For 56 days, the growth on the slants was examined for visible colonies. All positive media were
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examined by Ziehl–Neelsen and gram staining to confirm the presence of only acid–fast bacteria,
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and the colonies were subcultured onto trypticase soy agar II with 5% sheep blood (TSA II;
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Becton Dickinson) to check for contaminants.
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DNA extraction. For Ogawa slants, a sample of DNA was extracted from a loopful (3–mm3
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sphere) of bacterial colony. Bacterial cells were mechanically disrupted with glass beads. After
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phenol-chloroform treatment (DDH Mycobacteria; Kyokuto Pharmaceuticals), DNA in the
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aqueous phase was extracted and purified on a robotic liquid handler AGE-96 (Biotec, Tokyo,
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Japan) with magnetic silica particles (MagneSil Blood Genomic Max Yield System; Promega,
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Madison, WI). For the MGIT 960 system, a 4.0-ml aliquot of culture broth was centrifuged for
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10 min at 13,000×g. The pellet was extracted with the bacterial DNA/RNA extraction kit
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(MORA–EXTRACT; Kyokuto Pharmaceuticals). Bacterial cells were mechanically disrupted
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with zirconia beads, and phenol-chloroform treatment was performed, as recommended by the
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manufacturer. A 100-µl aliquot of TE buffer (10 mM Tris–HCl, pH 8.0, 0.1 mM EDTA) was
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added to the extracted DNA pellet, and DNA concentrations were determined using the
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PicoGreen system (Molecular Probes, Eugene, OR), as recommended by the manufacturer.
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Amplicor PCR. For MGIT-positive culture, an aliquot of each culture was tested in parallel by
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the Amplicor PCR (Roche Diagnostic Systems) for M. tuberculosis complex, M. avium and M.
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intracellurare, as described in the manufacturer’s instructions.
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DNA sequencing. The 25-µl reaction mixture contained ExTaq HS buffer (Takara Shuzo, Ohtsu,
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Japan) with 2.0 mM MgCl2, 200 µM of each of the deoxynucleoside triphosphates, 1.0 U of EX
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Taq HS DNA polymerase, 10 ng of template, 10 pmol of each of the primers SSU–bact–27f
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(5'–AGA GTT TGA TCM TGG CTC AG–3') and SSU–bact–907r (5'–CCG TCA ATT CMT TTR
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AGT TT–3') for 16S rDNA, and 16S–1511f (5'–AAG TCG TAA CAA GGT ARC CG–3') and
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23S–23r (5'–TCG CCA AGG CAT CCA CC–3') for ITS-1 region (18). Amplification was
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performed with GeneAmp PCR System 9700 ThermalCycler (Applied Biosystems) for 30 cycles
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(30 s at 94°C, 30 s at 53°C, and 90 s at 72°C), followed by an extension step at 72°C for 7 min.
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The PCR products were visualized with ethidium bromide staining and UV illumination.
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Purification of the amplicons was performed with the AMPure PCR Purification System
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(Agencourt, Beverly, MA), following the manufacturer’s instructions. The ABI Prism BigDye
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Terminator v1.1 Cycle Sequencing Ready Reaction kit (Applied Biosystems) was used for the
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sequencing of the PCR products. The sequencing reaction mixture contained 0.5 µl of Big Dye
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premix, 1.75 µl of 5×sequencing buffer, 1.6 pmol of sequencing primer, and approximately 10 ng
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of PCR product template in a total volume of 10 µl. SSU–bact–27f, SSU–bact–907r, 16S–1511f,
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and 23S–23r were used for sequencing primers for both DNA strands. The sequencing reaction
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was performed with a GeneAmp PCR system 9700 thermocycler (Applied Biosystems). A
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denaturation step at 95°C for 2 min proceeded 25 cycles (10 s at 96°C, 15 s at 53°C, and 2.5 min
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at 60°C). Sequencing products were purified with a CleanSEQ Sequencing Reaction Clean-Up
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system (Agencourt) and analyzed with the 3130xl Genetic Analyzer (Applied Biosystems),
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following the manufacturer’s instructions. Raw sequencing data were edited to resolve
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discrepancies by evaluating the electrophoretograms with the Sequencing Analysis software v
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3.3 (Applied Biosystems). The edited sequence data from both strands were aligned with the
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DNASIS Pro (Hitachi Software Engineering, Yokohama, Japan). We analyzed the consensus
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sequence of approximately 500 bp of the 5′–end of the 16S rDNA for comparison with the
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sequence databases stored in GenBank (BLAST; http://www.ncbi.nlm.nih.gov/BLAST/) or the
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Ribosomal Differentiation of Medical Microorganisms (RIDOM; http://www.ridom–rdna.de/) (9,
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10).
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Probe design for Invader assay. The 16S rDNA and ITS-1 sequences were aligned with those
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from the GenBank database and quality-controlled RIDOM database. With Invader Creator
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software (Third Wave Technologies), 20 specific probes were designed to identify and
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differentiate mycobacteria in conserved regions of ITS-1 and 16S rDNA hypervariable regions A
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and B, including species-specific sites. In addition, mixed GNS probes for the mycobacteria
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genus–specific region and BRB probe for the bacteria universal region were designed from
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conserved regions of 16S rDNA (Table 1). Signal probes and Invader oligonucleotide for the
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specific nucleotide sequences were designed to have theoretical annealing temperatures of 63°C
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and 77°C, respectively, using a nearest-neighbor algorithm on the basis of final probe and target
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concentrations. The signal probes and Invader oligonucleotides used to detect Mycobacterium
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species by the Invader assay are shown in Table 1.
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Biplex Invader assay. The Invader assay utilizes the thermostable flap endonuclease Cleavase
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XI, which cleaves invasive structures formed from single-base overlap between the Invader
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oligonucleotide and the signal probe when hybridized to a complementary target DNA. This
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method can accurately discriminate single-base differences, such as SNPs, and can measure
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genomic DNA (≥104 copy/assay). The Invader assay combines structure-specific cleavage
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enzymes and a universal FRET (Förster resonance energy transfer) system, and the biplex format
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of the Invader assay enables simultaneous detection of two kinds of species in a single well. At
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first, 3 µl of genomic DNA (0.03–0.33 ng/µl) was added into a 384-well plate, 6 µl of mineral oil
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(Sigma) was overlayed into all reaction wells, the mixtures were denatured by incubation at 95°C
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for 10 min, and 3 l of the appropriate reaction mixture was added. The reaction mixture
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contained 32 ng Cleavase XI enzyme, 0.817 µmol/l of each signal probe, 0.163 µmol/l of each
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Invader oligonucleotide, 0.65 µmol/l each of FAM dye and Redmond Red dye FRET cassettes
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(Epoch Bioscience, Redmond, WA), 5.04% PEG8000, 30.7 mmol/l MgCl2 and 24.5 mmol/l
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MOPS. After the reagent was dispensed, the plate was spun for 10 sec at 400×g, then
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sequentially incubated isothermally at 64°C up to 4 h in the thermal fluorescence microtiter plate
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reader (Fluodia; Otsuka Electronics, Osaka, Japan), while the fluorescent intensities were
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measured at 15 min intervals for FAM (excitation: 486 nm; emission: 530 nm) and Redmond
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Red (RED) (excitation: 560 nm; emission: 620 nm).
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Invader assay data analysis. Raw data were analyzed using a Microsoft Excel–based
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spreadsheet (Microsoft, Redmond, WA). For each specific signal, fold-over-zero (FOZ) values
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were calculated as follows for the signal obtained with each dye:
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FOZ = Raw counts from sample/ Raw counts from no target control.
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DNA samples from 54 mycobacterial reference strains were used to determine the cut-off value
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of FOZ. The mean + 5 S.D. of the FOZ value of non-target sequence in each well was calculated
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to 1.22–1.95 (probe set 1–10) of FAM-FOZ and 1.23–1.70 (probe set 1–10) of RED-FOZ.
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Therefore, the cut-off level was set at 2.00.
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RESULTS
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Probe design and validation
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The most widely accepted gene for bacterial identification is the 16S rDNA, but this gene alone
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lacks sufficient resolution to identify all mycobacterial species (5, 32, 37, 39). For example, the
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16S rDNA could not distinguish between M. kansasii and M. gastri, and also between M.
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chelonae and M. abscessus. However, since the ITS-1 region had greater diversity than the 16S
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rDNA (27, 18), the Invader probes designed in the ITS-1 region could distinguish these strains.
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Therefore, the Invader assay was set up with a more effective combination of the 16S rDNA and
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the ITS-1 region.
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To validate the Invader probes, we evaluated 65 type and 13 reference strains, and 607
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mycobacterial clinical strains. Specific signals were obtained for the target sequences of the type
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and reference strains, according to the criteria of the Invader assay. The GNS and the BRB
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probes were designed for the mycobacterial genus–specific region and the bacteria universal
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regions, respectively. Using the Invader assay, strains that reacted only to both the BRB and GNS
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probes were mycobacteria other than target species, and strains that reacted to only the BRB
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probe were bacteria other than mycobacteria (Table 2). Next, we confirmed the variation and
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conservation of target sequences in 607 clinical mycobacterial strains, which were identified by
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DNA sequencing of the 16S rDNA and ITS-1. For all 607 clinical strains, the identities
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determined by DNA sequencing corresponded exactly to those determined by the Invader assay
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(Table 3).
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Identification directly from the BACTEC MGIT samples. By examining a serial dilution of
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reference strains, the detection limit of genomic DNAs for all Invader probes was found to be
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0.03 ng/µl. DNA extracted from 122 positive samples of the MGIT 960 system had a minimum
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concentration of < 0.01 ng/µl (less than the lower limit of the PicoGreen system), a maximum
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value 24.38 ng/µl, and an average of 4.40 ng/µl. Zirconia beads efficiently extracted DNA for M.
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tuberculosis and NTM. Six MGIT samples were negative in the Invader assay, and the DNA
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concentrations of four MGIT samples were below the detection limit (< 0.01 ng/l). Although
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the remaining two MGIT samples had sufficient DNA for the Invader assay (from ≥ 1 ng/l to
