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

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

175 180 185 190 195 200

175 180 185 190 195 200 combinations and by using 10-fold dilutions of M. pneumoniae, C. pneumoniae and Legionella spp. as single targets in lysis buffer (Tables 4a, 4b and 5). The sensitivity was also studied using 10-fold dilutions of cultured M. pneumoniae, C. pneumoniae, and L. pneumophila serotype 1 added as a single target to protease treated samples (16) of each respiratory specimen pool. The 95% hitrate for in vitro generated RNA was calculated by SAS version 6.12 software (SAS, Cary, USA). Reproducibility The intrarun and interrun variations in multiplex real-time NASBA were estimated by running samples containing 500 or 5000 CCU/100 µl of M. pneumoniae; 0.1, 1, 10 or 100 IFU/100 µl of C. pneumoniae and of 100 or 1000 CFU/100 µl of L. pneumophila serotype 1 added as single targets in duplicate to 1 BAL pool to determine the intrarun variation and to 2 BAL pools to determine the interrun variation. Five replicates of each nucleic acid extract were analysed, respectively. The calculations on the final fluorescent value were done by using the Microsoft Excel software. Negative controls were co- analyzed within each run. ACCEPTED Serology and Legionella urinary antigen test Serology and urinary antigen tests (uAg) were performed on physician's request. If available, paired sera were tested. For the detection of M. pneumoniae antibodies, an IgM and an IgG enzyme immunoassay (Anilabsystems, Helsinki, Finland) were performed. An acute infection was defined when at least a 1.5-fold IgG EIU increase with paired sera, assayed in the same run was obtained. With EIU values above 130, a 1.3-fold increase in the same run was indicative of a significant rise in antibodies. IgM was considered positive when the signal/cut-off units were above 1.1. C. pneumoniae specific IgM and IgG antibodies were detected by the Anilabsystems EIA. The same definition of an acute infection as described above was used. A patient was considered positive for Legionella when >70U/ml and/or >140 U/ml were measured for IgG and IgM, respectively, by using the Serion ELISA classic 8

205 210 215 220 225 (Virion/Serion, Würzburg, Germany) IgM and IgG test. The BINAX NOW urinary antigen test was used according to the instructions of the manufacturer. Statistical analysis: The Х 2 -test was used for statistical analysis. Results Specificity of the 16S rRNA multiplex NASBA primers Using real-time multiplex NASBA, positive results were obtained with nucleic acid extracts from M. pneumoniae, C. pneumoniae, and all Legionella spp and serotypes but with none of the other organisms listed in Table 1. Sensitivity of the 16S rRNA multiplex NASBA Single target: ACCEPTED The 95% hit-rate for the analytical sensitivity of the 16S rRNA NASBA primers tested on dilutions of in vitro generated WT RNA was 33 molecules, 153 molecules and 169 molecules of C. pneumoniae, M. pneumoniae and L. pneumophila in vitro generated RNA, respectively, when immediately added to the amplification reactions. When extraction was done prior to the amplification, i.e. the isolation of in vitro generated RNA from lysis buffer, 3185 molecules, 18489 molecules and 16697 molecules of C. pneumoniae, M. pneumoniae and L. pneumophila, respectively, were needed in the extraction for a 95% hit-rate in the amplification reaction. However, it should be mentioned that only 10% of the extracted nucleic acid is used in the amplification reaction. The results for the individual reactions are shown in Tables 4a and 4b. When lysis buffer was spiked with 5 CCU/100 µl of M. pneumoniae, 4/4 and 2/4 samples were found positive by real-time mono and multiplex NASBA, respectively. With an input of 0.1 IFU/100µl, of C. pneumoniae 4/4 and 3/4 samples were positive by real-time mono and multiplex NASBA respectively. Real-time mono and multiplex NASBA 9

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