This Article
Right arrow Full Text
Right arrow Full Text (PDF)
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 Dingle, K. E.
Right arrow Articles by Jeffery, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dingle, K. E.
Right arrow Articles by Jeffery, K.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, March 2004, p. 1003-1011, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1003-1011.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Stable and Noncompetitive RNA Internal Control for Routine Clinical Diagnostic Reverse Transcription-PCR

Kate E. Dingle,1,2* Derrick Crook,1,2 and Katie Jeffery2

Nuffield Department of Clinical Sciences, Oxford University,1 Department of Microbiology, Oxford Radcliffe NHS Trust, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom2

Received 22 September 2003/ Returned for modification 13 November 2003/ Accepted 9 December 2003

Clinical diagnostic tests based on nucleic acid amplification assist with the prompt diagnosis of microbial infections because of their speeds and extremely low limits of detection. However, the design of appropriate internal controls for such assays has proven difficult. We describe a reaction-specific RNA internal control for diagnostic reverse transcription (RT)-PCR which allows extraction, RT, amplification, and detection to be monitored. The control consists of a G+C-rich (60%) RNA molecule with an extensive secondary structure, based on a modified hepatitis delta virus genome. The rod-like structure of this RNA, with 70% intramolecular base pairing, provides a difficult template for RT-PCR. This ensures that the more favorable target virus amplicon is generated in preference to the control, with the control being detected only if the target virus is absent. The unusual structure of hepatitis delta virus RNA has previously been shown to enhance its stability and resistance to nucleases, an advantage for routine use as an internal control. The control was implemented in three nested multiplex RT-PCRs to detect nine clinically important respiratory viruses: (i) influenza A and B viruses, (ii) respiratory syncytial viruses A and B and human metapneumovirus, and (iii) parainfluenza virus types 1 to 4. The detection limits of these assays were not detectably compromised by the presence of the RNA control. During routine testing of 324 consecutive unselected respiratory samples, the presence of the internal control ensured that genuine and false-negative results were distinguishable, thus increasing the diagnostic confidence in the assay.

* Corresponding author. Mailing address: Microbiology Level 6, Oxford University, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, United Kingdom. Phone: 44 1865 220870. Fax: 44 1865 764192. E-mail: kate.dingle{at}ndcls.ox.ac.uk.

Journal of Clinical Microbiology, March 2004, p. 1003-1011, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1003-1011.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Picard, F. J., Gagnon, M., Bernier, M. R., Parham, N. J., Bastien, M., Boissinot, M., Peytavi, R., Bergeron, M. G. (2009). Internal Control for Nucleic Acid Testing Based on the Use of Purified Bacillus atrophaeus subsp. globigii Spores. J. Clin. Microbiol. 47: 751-757 [Abstract] [Full Text]  
  • Villanova, G. V., Gardiol, D., Taborda, M. A., Reggiardo, V., Tanno, H., Rivadeneira, E. D., Perez, G. R., Giri, A. A. (2007). Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR. J. Clin. Microbiol. 45: 3555-3563 [Abstract] [Full Text]  
  • Letant, S. E., Ortiz, J. I., Bentley Tammero, L. F., Birch, J. M., Derlet, R. W., Cohen, S., Manning, D., McBride, M. T. (2007). Multiplexed Reverse Transcriptase PCR Assay for Identification of Viral Respiratory Pathogens at the Point of Care. J. Clin. Microbiol. 45: 3498-3505 [Abstract] [Full Text]  
  • Harnden, A., Perera, R., Brueggemann, A. B, Mayon-White, R., Crook, D. W, Thomson, A., Mant, D. (2007). Respiratory infections for which general practitioners consider prescribing an antibiotic: a prospective study. Arch. Dis. Child. 92: 594-597 [Abstract] [Full Text]  
  • Das, A., Spackman, E., Senne, D., Pedersen, J., Suarez, D. L. (2006). Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents.. J. Clin. Microbiol. 44: 3065-3073 [Abstract] [Full Text]  
  • Huang, Q., Cheng, Y., Guo, Q., Li, Q. (2006). Preparation of a chimeric armored RNA as a versatile calibrator for multiple virus assays.. Clin. Chem. 52: 1446-1448 [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»