Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

Journal of Clinical Microbiology
K LoensH Goossens

Abstract

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 10(4) molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results i...Continue Reading

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Citations

May 28, 2011·Neuropathology : Official Journal of the Japanese Society of Neuropathology·Alexis SimpkinsSuash Sharma
Nov 8, 2003·Journal of Clinical Microbiology·K LoensM Ieven
Sep 6, 2003·Journal of Clinical Microbiology·K LoensH Goossens
Feb 11, 2010·Future Microbiology·Patrick Tang, Charles Chiu
Jun 12, 2013·Nanomedicine : Nanotechnology, Biology, and Medicine·Wen-Hsin ChangGwo-Bin Lee
Jan 15, 2011·Molecular and Cellular Probes·Dmitriy V VolokhovVladimir E Chizhikov
Mar 7, 2007·Diagnostic Microbiology and Infectious Disease·Tânia Sueli de AndradeRosario Dominguez Crespo Hirata
Aug 30, 2008·FEMS Microbiology Reviews·Thomas Prescott AtkinsonKen B Waites
May 11, 2007·Expert Review of Molecular Diagnostics·Miriam WittekAnnemarie Berger
Jan 26, 2006·Journal of Medical Microbiology·David PitcherTimothy G Harrison
Feb 15, 2013·Lab on a Chip·Chen-Min ChangGwo-Bin Lee
Aug 18, 2006·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Xiao-Song WangWei-Feng Chen
Nov 17, 2020·Frontiers in Cellular and Infection Microbiology·Fei ZhaoJianzhong Zhang

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