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Development of Real-Time Multiplex NASBA® for the - Journal of ...

Development of Real-Time Multiplex NASBA® for the - Journal of ...

65 70 75 80 85 90

65 70 75 80 85 90 Bacterial strains 1.2 Materials and Methods Bacterial strains, used to test the specificity of the NASBA® primers, are presented in Table 1. Mycoplasma strains were cultured in homemade spiroplasma (SP4) medium (28) without thallium acetate and supplemented with amphotericin B (0.5 mg/ml), polymyxin B (500 U/ml), glucose (0.5 %), and arginine (0.25 %) or urea (0.5 %) depending on the nutritional needs of the species. M. pneumoniae strain ATCC 29085 was quantified by incubation of 4 replicates of 10- fold dilutions of a suspension in SP4 medium at 37°C. The cultures were examined weekly for color change from red to yellow during 2 months. The titer was expressed in color changing units (CCU) per ml (3). C. pneumoniae (ATCC VR-1355) was grown in Hep-2 cells. After inoculation, the cell cultures were centrifuged at 3500 rpm and 25°C for 60 min and subsequently incubated at 37°C for 1h. Then the medium was aspirated and cell cultures were incubated with fresh ACCEPTED medium containing cycloheximide (1mg/l). After 3 days, the shell vials were aspirated and fixed with 96% ethanol. The fixed monolayers were rinsed with phosphate buffered saline and stained with fluorescent antibody technique with C. pneumoniae specific mouse monoclonal antibodies (Dako A/S, Glostrup, Denmark). Rabbit anti-mouse immunoglobulin labeled with fluoresceine isothiocyanate (Dako A/S) was used as a conjugate. C. pneumoniae (ATCC VR-1355) was quantified by incubation of 5 replicates of 10-fold dilutions on Hep-2 cells. Inclusion forming units (IFU) were counted 72h after infection by immunofluorescence microscopy. The titer was expressed in IFU/ml. Legionella strains were grown on buffered charcoal-yeast (Oxoid Ltd., Belgium) agar plates at 37°C for 48-72h and quantified by incubation of 4 replicates of 10-fold dilutions on buffered charcoal-yeast agar. The titer was expressed as CFU/ml. The other organisms were cultivated on standard media supporting optimal growth. The clinical isolates were identified by standard methods. 4

95 100 105 110 115 Respiratory specimens Throat swabs, broncho-alveolar lavages (BAL), nasopharyngeal aspirates (NPA), sputum and bronchus aspirates (BA) from the Microbiology Laboratory of the University Hospital of Antwerp and tested negative for M. pneumoniae, C. pneumoniae and L. pneumophila by PCR (12, 29) were spiked with dilutions of reference strains for sensitivity experiments. Archived specimens positive by PCR for one of the three organisms: 3 C. pneumoniae positive nasopharyngeal aspirates, 5 L. pneumophila positive lung biopsy specimens (Table 2b), 4 sputum specimens and 5 water samples. Fifty-one respiratory specimens were collected from 33 patients found to be M. pneumoniae positive by PCR (12). Forty of these specimens were M. pneumoniae PCR positive (Table 2). One hundred forty nine specimens (44 sputum specimens, 36 throat swabs, 25 NPA, 22 BA, 19 BAL, 1 gargle specimen, 1 lung biopsy specimen and 1 pleural fluid) previously found negative for M. pneumoniae, C. pneumoniae and L. pneumophila by PCR were also tested as negative control specimens. Nucleic acid extraction ACCEPTED All respiratory specimens were protease treated before extraction (16). Nucleic acids were extracted as described by Boom et al. (5) using the NucliSens Basic Kit module (bioMérieux, Boxtel, The Netherlands). Briefly, 100 µl of protease treated clinical specimens, (16) or aliquots of a bacterial culture, were added to a guanidinium thiocyanate (GuSCN) lysis solution, pH 6.2 and mixed vigorously for rapid lysis. Fifty µl of activated silica were added. The nucleic acid-silica complex was washed twice with GuSCN washing solution, twice with 70% (v/v) ethanol and once with acetone. After drying at 56°C, nucleic acids were eluted from the silica using 50µl elution buffer and stored at -80°C. Each nucleic acid extract was amplified by both real-time mono NASBA and by the real-time multiplex NASBA. 5

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