HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Nolte, F. S. Search for Related Content PubMed PubMed Citation Articles by Nolte, F. S. Related Collections Molecular Diagnostics and Genetics Laboratory Management Hemostasis and Thrombosis Endocrinology and Metabolism

Novel Internal Controls For Real-Time PCR Assays

¹ Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, E-mail fnolte@emory.edu

The first 20% of the full text of this article appears below.

In the 19 years since the first descriptions of the PCR (1), nucleic acid amplification methods have made the transition from research to clinical laboratories. Molecular diagnostics are now firmly established as part of laboratory medicine, with applications in genetics, oncology, pharmacology, and infectious disease. Routine diagnostic applications of these methods have been made possible by the thoughtful use of controls coupled with laboratory practices intended to reduce false-positive and -negative results (2)(3)(4).

Nucleic acid amplification assays are prone to inhibition by a variety of substances found in clinical samples. Although the identities and biochemical mechanisms of action of many inhibitors remain unclear, bile salts and complex polysaccharides in feces (5), heme in blood (6), and urea in urine(7) have all been shown to inhibit PCR, probably through interference with the binding and/or polymerization activity of DNA polymerases. Carryover of reagents used for isolation of nucleic acids from clinical specimens can also inhibit amplification reactions. Other causes of false-negative results include target nucleic acid degradation, sample processing errors, thermal cycler malfunction, and in reverse transcription-PCR, failure of the reverse transcription step.

A simple approach to detect inhibitors is to add the target nucleic acid to a separate aliquot of the sample after processing. Addition of the external control to a separate reaction, however, doubles the cost of the assay, and the approach may be unworkable if batch sizes are large. Moreover, addition of the control after sample processing . . . [Full Text of this Article]

The following articles in journals at HighWire Press have cited this article:

				R. van Doorn, M. M. Klerks, M. P. E. van Gent-Pelzer, A. G. C. L. Speksnijder, G. A. Kowalchuk, and C. D. Schoen Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays Appl. Envir. Microbiol., November 15, 2009; 75(22): 7253 - 7260. [Abstract] [Full Text] [PDF]

				Q. Huang, Y. Cheng, Q. Guo, and Q. Li Preparation of a chimeric armored RNA as a versatile calibrator for multiple virus assays. Clin. Chem., July 1, 2006; 52(7): 1446 - 1448. [Full Text] [PDF]

				C. Drosten, M. Panning, J. F. Drexler, F. Hansel, C. Pedroso, J. Yeats, L. K. de Souza Luna, M. Samuel, B. Liedigk, U. Lippert, et al. Ultrasensitive Monitoring of HIV-1 Viral Load by a Low-Cost Real-Time Reverse Transcription-PCR Assay with Internal Control for the 5' Long Terminal Repeat Domain Clin. Chem., July 1, 2006; 52(7): 1258 - 1266. [Abstract] [Full Text] [PDF]

				S. Burggraf, U. Reischl, N. Malik, M. Bollwein, L. Naumann, and B. Olgemoller Comparison of an Internally Controlled, Large-Volume LightCycler Assay for Detection of Mycobacterium tuberculosis in Clinical Samples with the COBAS AMPLICOR Assay J. Clin. Microbiol., April 1, 2005; 43(4): 1564 - 1569. [Abstract] [Full Text] [PDF]

				L. L. M. Poon, C. S. W. Leung, K. H. Chan, J. H. C. Lee, K. Y. Yuen, Y. Guan, and J. S. M. Peiris Detection of Human Influenza A Viruses by Loop-Mediated Isothermal Amplification J. Clin. Microbiol., January 1, 2005; 43(1): 427 - 430. [Abstract] [Full Text] [PDF]