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Jérôme Mounier,Geneviève Héry-Arnaud,Audrey Gouëllo,Marlène Keravec,Solène Le Gal,Grégory Pacini,Stella Debaets,Gilles Nevez,Gilles Rault,Georges Barbier 한국미생물학회 2014 The journal of microbiology Vol.52 No.4
The aim of this study was to evaluate the use of denaturinghigh-performance liquid chromatography (DHPLC) to characterizecystic fibrosis (CF) airway microbiota includingboth bacteria and fungi. DHPLC conditions were first optimizedusing a mixture of V6, V7 and V8 region 16S rRNAgene PCR amplicons from 18 bacterial species commonlyfound in CF patients. Then, the microbial diversity of 4 sputumsamples from 4 CF patients was analyzed using culturalmethods, cloning/sequencing (for bacteria only) and DHPLCpeak fraction collection/sequencing. DHPLC analysis allowedidentifying more bacterial and fungal species than the classicalculture methods, including well-recognized pathogenssuch as Pseudomonas aeruginosa. Even if a lower number ofbacterial Operational Taxonomic Units (OTUs) was identifiedby DHPLC, it allowed to find OTUs unidentified bycloning/sequencing. The combination of both techniquespermitted to correlate the majority of DHPLC peaks to definedOTUs. Finally, although Aspergillus fumigatus detectionusing DHPLC can still be improved, this techniqueclearly allowed to identify a higher number of fungal speciesversus classical culture-based methods. To conclude, DHPLCprovided meaningful additional data concerning pathogenicbacteria and fungi as well as fastidious microorganisms presentwithin the CF respiratory tract. DHPLC can be consideredas a complementary technique to culture-dependentanalyses in routine microbiological laboratories.