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

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

15 20 25 30 35 Abstract

15 20 25 30 35 Abstract Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae and L. pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild type in vitro generated RNA in water, dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae, C. pneumoniae and L. pneumophila positive and negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the mono real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive ACCEPTED than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, and C. pneumoniae and Legionella spp. in respiratory specimens, regarding handling, speed and number of samples that can be analyzed in a single run. 1.1 Introduction Community-acquired pneumonia is associated with significant morbidity, mortality and utilization of health service resources. No etiologic agent can be identified in >35% of cases of CAP when using conventional diagnostic methods such as culture and serology (21, 22, 31). M. pneumoniae, C. pneumoniae and L. pneumophila may be responsible for 15-50% of CAP (23, 25). Differentiation of infections due to S. pneumoniae from those due to M. pneumoniae, C. pneumoniae or L. pneumophila as well as those due to viruses is essential to avoid inappropriate use of antibiotics. 2

40 45 50 55 60 Culture and serological confirmation of the diagnosis of infections due to M. pneumoniae, C. pneumoniae and L. pneumophila is difficult and may require several weeks. Therefore, nucleic acid amplification techniques are of considerable interest. PCR was shown to be significantly more sensitive than culture for the detection of M. pneumoniae (1, 33), C. pneumoniae (4, 32), and L. pneumophila (2, 11). Nucleic acid sequence-based amplification (NASBA, bioMérieux, Boxtel, The Netherlands) targeted at RNA has been adapted to the real-time format using DNA hybridization probes that fluoresce upon hybridization (15). The whole process of amplification and detection runs in a fluorescent reader. Real-time assays enable one-tube assays suitable for high-throughput applications, reducing the assay time and limiting potential contamination between samples. Real-time single-target (mono) NASBA has been successfully used for the identification of West Nile and St. Louis encephalitis viruses (14), human immunodeficiency virus type 1 (8), M. pneumoniae (18), and Clavibacter michiganensis subsp. sepedonicus (30). Multiplex formats might solve the practical shortcoming of detecting only one agent at a time. Detection of multiple pathogens simultaneously would be economical for small volume samples and reduce costs. Multiplex real-time NASBA has already been ACCEPTED successfully applied for the detection of potato leafroll virus and potato virus Y in potato tubers (13), for simultaneous detection and typing of potato virus Y isolates (26) and monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA (9). The aim of this study was to develop a real-time multiplex NASBA assay for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens based on the amplification of a 16S rRNA target sequence using the NucliSens Basic Kit (17) and to compare the results with those of real-time NASBA. 3

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