Development of a Duplex Immunocapture Reverse-Transcription Quantitative Polymerase Chain Reaction for Large-Scale Detection of Potato Mop-Top Virus in Dormant Potato Tubers.

Abstract

Potato is an important sector to the U.S. economy, and it created over $100 billion in economic activity in 2021. The U.S. exports fresh potatoes to several countries. In certain cases, fresh potato shipments from the U.S. to the international market requires the crop to be free of potato mop-top virus (PMTV). This increased the need to provide potato growers with an optional, reliable and large-scale detection method of PMTV, especially in asymptomatic tubers. We developed a duplex Immunocapture Reverse-Transcription Quantitative Polymerase Chain Reaction (IC-RT-qPCR) for the large-scale detection of PMTV in dormant tubers. The IC step eliminates the need for RNA extraction kits, making this assay appropriate for large scale tuber testing. To enhance the reliability of the current assay and reduce the chance of false negatives, a duplex format was used by deploying two primer-probe sets, including a previously reported primer-probe set targeting the RNA-CP, and a newly designed primer-probe set targeting a conserved region of RNA-TGB of PMTV genome. We also determined that peels from the stem end of the tubers were more likely to test positive for PMTV than bud end peels or lateral tuber cores. The duplex IC-RT-qPCR will provide a reliable and sensitive tool for the large-scale detection of PMTV in dormant tubers and will help safeguard potato movement in the U.S. and internationally.