A sensitive batch detection of banana bunchy top virus using SYBR^® Green real-time PCR

Banana bunchy top virus (BBTV) is the most destructive viral disease of banana crop in the Philippines. The disease causes heavy damage to important local varieties, ‘Lakatan’ and ‘Cavendish’. Infected planting materials can cause long-term disease transmission causing geographical location to dictate genetic variation among viral strains. Hence, there is a need for an efficient and reliable quarantine detection procedure. This study developed a high-throughput real-time PCR protocol for batch detection of BBTV. A primer set derived from the DNA-R region of the virus was designed for specific BBTV detection. Tests for optimal annealing temperature, sample load, and sensitivity were performed. Finally, the cost per sample was compared to conventional end-point PCR. Optimization of the annealing temperature, from 55.5 ℃ to 63.5 ℃, yielded virus detection. The detection protocol developed was efficient to detect BBTV from a leaf disc measuring up to 5 mm diameter and weight of approximately 3 mg. DNA from infected leaf discs was detectable up to 1:10000 dilution. Sample pooling was detectable up to 1:99 infected to healthy leaf disc ratio. This sensitive and cost-efficient batch detection method for BBTV detection will be useful for quarantine services and various diagnostic applications.