Multiplex Reverse Transcription (RT) High-Fidelity PCR Protocol for the Detection of Six Viruses that Cause Potato Tuber Necrosis

Viruses that cause necrotic symptoms in potato tubers can be difficult to distinguish based on symptoms and frequently require multiple molecular tests to identify the pathogen. In this study, a multiplex RT PCR high fidelity PCR protocol was developed using previously validated primers that could accurately detect six important potato viruses and detect multiple viruses in a single sample simultaneously. To test the protocol, 53 tubers previously tested using conventional PCR were retested using this multiplex protocol and blindly evaluated. In every case, the multiplex PCR results were consistent with previous findings. In addition, the multiplex PCR protocol also detected several mixed infections that were previously undetected. The results demonstrate that this technique could be a valuable assay not only for diagnostic laboratories, but also for seed certification and regulatory agency laboratories to diagnose the virus(es) that may be present in tubers with surface or internal necrotic symptoms.