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Journal of Clinical Microbiology, May 2003, p. 2055-2061, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2055-2061.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Applications of Competitor RNA in Diagnostic Reverse Transcription-PCR

Steven B. Kleiboeker*

Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211

Received 4 November 2002/ Returned for modification 8 February 2003/ Accepted 20 February 2003

Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

* Mailing address: Department of Veterinary Pathobiology and the Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, 1600 E. Rollins, Columbia, MO 65211. Phone: (573) 882-6811. Fax: (573) 882-1411. E-mail: KleiboekerS{at}Missouri.edu.

Journal of Clinical Microbiology, May 2003, p. 2055-2061, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2055-2061.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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