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

摘要

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 (R) 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 (R) PCR system was highly sensitive, detecting as few as 2.76 copies of BBTV genomic DNA or 1.0 ng-1.0 mg of infected banana leaves. The entire assay could be completed within 2 h. Regression analysis showed that the quantitative results of TaqMan (R) 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