This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodríguez-Lázaro, D.
Right arrow Articles by Cook, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodríguez-Lázaro, D.
Right arrow Articles by Cook, N.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 2004, p. 5832-5836, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5832-5836.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

David Rodríguez-Lázaro,1 Martin D'Agostino,2 Maria Pla,1* and Nigel Cook2

Institute of Food and Agricultural Technology, University of Girona, Girona, Spain,1 Defra Central Science Laboratory, Sand Hutton, York, United Kingdom2

Received 2 December 2003/ Returned for modification 2 February 2004/ Accepted 25 August 2004

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5' end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results.

* Corresponding author. Mailing address: Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi, s/n 17071 Girona, Spain. Phone: 34 972 419852. Fax: 34 972 418399. E-mail: maria.pla{at}udg.es.

Journal of Clinical Microbiology, December 2004, p. 5832-5836, Vol. 42, No. 12
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.12.5832-5836.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lopez-Enriquez, L., Rodriguez-Lazaro, D., Hernandez, M. (2007). Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR. Appl. Environ. Microbiol. 73: 3747-3751 [Abstract] [Full Text]  
  • Fenicia, L., Anniballi, F., De Medici, D., Delibato, E., Aureli, P. (2007). SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A. Appl. Environ. Microbiol. 73: 2891-2896 [Abstract] [Full Text]  
  • Rodriguez-Lazaro, D., Pla, M., Scortti, M., Monzo, H. J., Vazquez-Boland, J. A. (2005). A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control. Appl. Environ. Microbiol. 71: 9008-9012 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»