Developing a reliable assay for Cacao Swollen Shoot Virus using quantitative PCR

Cacao Swollen Shoot Virus (CSSV) causes a devastating disease in Theobroma cacao, the main economic crop in West Africa. The CSSV genome consists of a 7.4 Kb circular double-stranded DNA molecule that encodes a reverse transcriptase. CSSV does not functionally integrate into the genome of the host and requires an RNA transcript phase for infection. Because infected trees are asymptomatic for a long period, development of a sensitive method for CSSV detection is critical to maintain healthy cacao germplasm and particularly important for international germplasm exchange. Using the most conserved regions of six published CSSV genome sequences, we have developed quantitative PCR primers for both CSSV DNA and RNA detection. Quantitative PCR analysis with primers designed to various regions of the genome showed no detectable signals in DNA extracted from leaves of healthy plants. DNA extracted from infected trees gave strong CSSV positive signals for half of the samples tested.  A 10-fold increase in template amount in the qPCR assay gave similar results, with only half of the infected samples showing CSSV positive signals.  Progress and challenges to CSSV detection with quantitative PCR will be discussed.