Title: An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y Authors
|Ali, Mohamad Chikh -|
|Karasev, Alex -|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 4, 2013
Publication Date: April 17, 2013
Citation: Ali, M., Gray, S.M., Karasev, A. 2013. An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y. Plant Disease. doi: 10.1094/PDIS-02-13-0161-SR. Interpretive Summary: The number of strains of Potato virus Y that emerged recently in the United States and that are affecting the potato crop has increased drastically and the diagnostic methods to correctly identify and differentiate the virus strains has lagged. Several of the new strains have the potential to cause significant yield and quality losses to both the seed and commercial potato industries. It is important that seed lots infected with this virus be identified and eliminated from seed stocks before they can be planted. The detection and diagnosis of PVY strains is currently accomplished using a combination of several diagnostic methods because no one technique can correctly identify all the emergent strains. Here we report on the ability of a new diagnostic assay that is able to utilize differences in the genome sequence of nine of the 10 virus strains that occur in the U.S. The assay when combined with a widely used serological assay can correctly diagnose all PVY strains currently known to infect potato in the U.S. This is valuable information for diagnostic, regulatory and seed certification laboratories that need to quickly and accurately identify the PVY strains that will have the greatest impact on the potato crop.
Technical Abstract: A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed for the first time that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi and PVYN:O ,which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.