Duplex reverse transcription-polymerase chain reaction system to detect Potato spindle tuber viroid using an internal control mRNA and a non-infectious positive control RNA

An accurate and reliable system to detect Potato spindle tuber viroid (PSTVd) was developed based on duplex reverse transcription-polymerase chain reaction (RT-PCR) in combination with a plant mRNA internal control to evaluate the quality of RNA extracted from a dormant potato tuber and the effectiveness of the RT-PCR. In addition, a non-infectious RNA based on the PSTVd sequence was generated in vitro as a readily available positive control without the possibility of escape; this RNA was distinguishable from true positives in PSTVd infection. This detection system will be useful in plant quarantine and phytosanitary certification applications.