A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection

Abstract

A highly sensitive assay, based on polymerase chain reaction amplification of cDNA synthesized from the viral RNA of antibody-captured viral particles, has been developed for plum pox potyvirus (PPV) detection. The reaction, called immunocapture/PCR (IC/PCR), yields a specific 243-bp product. The immunocapture step, by allowing the use of large sample volumes and by the viral particle prepurification it achieves, dramatically increases the sensitivity of the assay. As few as 8000 target viral particles per ml of plant extract could be detected by IC/PCR. When compared to direct PCR (Wetzel et al., 1991), molecular hybridization using 32P-labeled cRNA probes and ELISA, this result corresponds to a 250-fold, 625-fold and 5000-fold increased sensitivity, respectively. The high sensitivity of IC/PCR was confirmed during an indexing trial with field samples collected from naturally infected trees. This very powerful technique should have wide ranging applications for the detection of a number of other viruses and pathogens for which specific antisera and sequence data are available.