A New Approach for the Rapid Detection and Genetic Discrimination of Mild and Severe Isolates of Citrus tristeza virus Based on ELISA/CE-SSCP

Abstract

A new approach for the rapid discrimination of mild and severe Citrus tristeza virus (CTV) isolates was developed by a sequential process made up of the DAS-ELISA immunological test followed by fluorescence-based Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP) analysis. The new method helps both in locating CTV infected trees and in preliminarily typing of the virus isolates, thus saving both time and resources. Partial p18 gene amplification products of CTV-RNAs directly extracted from infected leaves or recovered from ELISA plates are presented. Specific profiles of forward and reverse strands were obtained when biologically distinct CTV isolates were analysed both directly and following ELISA. The CE-SSCP method is simple, highly sensitive and highly reproducible, and can process a large number of samples for a variety of needs. Applied in a combined process, ELISA and CE-SSCP helped simultaneously to detect the virus and to collect useful information on the genetic diversity and structure of the CTV population present in a territory.