Applications of competitor RNA in diagnostic reverse transcription-PCR.
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.
Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA. KleiboekerS@Missouri.edu
Steven B
SB
Kleiboeker
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000235:genetics; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D034421:Sus scrofa; D014773:Virology / Q000379:methods; D014901:West Nile Fever / Q000175:diagnosis / Q000662:veterinary; D014902:West Nile virus / Q000235:genetics
D017931:DNA Primers; D012367:RNA, Viral