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.
Kleiboeker
Steven B
SB
Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA. KleiboekerS@Missouri.edu
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