High-throughput detection of banana bunchy top virus in banana plants and aphids using real-time TaqMan(®) PCR.

Banana bunchy top virus (BBTV) is the causal agent of banana bunchy top disease. Current diagnostic methods for BBTV are laborious and prone to generate false-negative results. A simple, reliable, and high-throughput method for detecting BBTV in plants and aphids has been developed, which involves tissues disruption from banana plants and viruliferous aphids followed by real-time TaqMan(®) PCR. Extraction of BBTV single-stranded DNA using this method is simple and less prone to contamination than using the CTAB (hexadecyltrimethylammonium bromide) method. The high throughput TaqMan(®) PCR system was highly sensitive, detecting as few as 2.76 copies of BBTV genomic DNA or 1.0 ng-1.0mg of infected banana leaves. The entire assay could be completed within 2h. Regression analysis showed that the quantitative results of TaqMan(®) PCR and copies of the virus have good correlation for plasmids (R(2)=0.966) and for infected leaves (R(2)=0.979). The method developed in this study can quantify BBTV in aphids and plants, even before the appearance of symptoms of banana bunchy top disease.