Poster: Diseases of Plants: Disease Detection & Diagnosis
Development and validation of a quadruplex real-time RT-PCR assay for simultaneous detection of three Citrus leprosis viruses in plants
G. WEI (1), A. Roy (2), W. Schneider (3), R. Brlansky (2), M. Nakhla (1) (1) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.; (2) University of Florida, IFAS, Plant Pathology Department, Citrus Research and Education Center, U.S.A.; (3) USDA-ARS-FDWSRU, U.S.A.
Citrus leprosis virus (CiLV) is the causal agent of Citrus leprosis - one of the most destructive diseases of citrus. CiLV is an RNA virus transmitted by Brevipalpus mite species. There are two major types of CiLV being described – one is the cytoplasmic type residing in the cytoplasm of infected cells and the other is the nuclear type primarily residing in the nucleus of infected cells. In the last decade CiLV spread swiftly from South America to Central America and recently has been detected in Mexico. The proximity and the potential threat to the multi-billion dollar US citrus industry have caused concerns in the United States where the disease has not been found since the 1960s. Molecular identification and early detection of each the cytoplasmic and nuclear types are essential to prevent CiLV from introduction and establishment in the US. In order to reduce testing time, cost and labor, we are developing and validating a quadruplex real-time RT-PCR assay for simultaneous detection of cytoplasmic CiLV-C, CiLV-C2, and nuclear CiLV-N with a Nad5 plant RNA internal control. From our repeated experiments, the results showed that sensitivity and specificity for the quadruplex RT-PCR assay were comparable to the level of monoplex RT-PCR detecting CiLV-C, CiLV-C2, and CiLV-N respectively. Further validation for the quadruplex RT-PCR assay including tests with additional field samples is in progress.