Detection and Differentiation of Potato Virus Y Strains from Potato Using Immunocapture Multiplex RT-PCR

摘要

Potato virus Y (PVY) is a serious problem for the seed potato industry in the United States. The maximum allowable infection level of PVY in certified seed potatoes is 2 % and is substantially lower in early generations of seed production. Moreover, recent emergence of genetically recombinant and serologically different strains of Potato virus Y has led to the development of diagnostic procedures to determine strain identity and to detect mixed strain infections more easily, sensitively and accurately. In the studies reported here a protocol for the detection of single or mixed PVY infections in potato incorporates the advantages of enzyme-linked immunoassay (ELISA) and multiplex reverse transcriptase PCR (RT-PCR). The viral particles from plant sap were enriched by ELISA and then lysed by heating to release the viral RNA for the reverse transcriptase. The cDNA product was used as template for the detection of infection by multiplex PCR eliminating the need for RNA extraction and handling. The sensitivity tests conducted indicate that the immunocapture reverse transcriptase PCR was more sensitive in detecting PVY in infected plant sap than multiplex RT-PCR or ELISA alone while retaining the ability to differentiate strains of PVY that can infect potato in the United States. The immunocapture multiplex RT-PCR described will be particularly useful for seed certification and diagnostic laboratories as a confirmatory test in conjunction with ELISA.