Immunocapture Polymerase Chain Reaction for the Detection and Characterization of Cacao Swollen Shoot Virus 1 A Isolates

Abstract

Abstract Oligonucleotides derived from the flanking regions of the putative coat protein gene of the cacao swollen shoot badnavirus isolate I A (CSSV‐1 A) were able to prime the synthesis of specific products directly from extracts from CSSV‐IA‐infected leaves by immunocapture polymerase chain reaction (IC‐PCR), following trapping of virions with polyclonal antibodies to CSSV‐1 A. CSSV isolates serologically distinct from CSSV‐I A were not detected by IC‐PCR when the CSSV‐I A‐derived primers were used following trapping with homologous antisera. IC‐PCR was at least 100‐fold more sensitive than double antibody sandwich (DAS)‐ELISA in comparative tests on samples from greenhouse‐grown cacao plants. The superior sensitivity of IC‐PCR over DAS‐ELISA was confirmed in attempts to detect and identify CSSV‐I A isolates in field samples and permitted detection of CSSV‐I A isolates even in symptomless leaves from plants showing stem swelling only. The IC‐PCR products obtained from four randomly selected field samples were sequenced and shown to contain a region of the CSSV‐I A genome where ORF X overlaps ORF 3. Analysis of the partial amino acid sequences deduced from ORF 3 and ORF X of the four field isolates revealed a considerable variation in these CSSV‐I A gene products.