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The Development of Three Strand Displacement Amplification Assays


									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

                                                  BD Biosciences • 54 Loveton Circle • Sparks, MD, USA 21152

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.
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 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: //
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
  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
                                                                                                                      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.

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.

    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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