title: High-throughput detection of banana bunchy top virus in banana plants and aphids using real-time TaqMan(®) PCR.
abstract: 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.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, No 1 West Beichen Road, Chaoyang District, Beijing 100101, China. cheny@im.ac.cn
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Hu
mesh_terms: D000818:Animals; D001042:Aphids / Q000821:virology*; D052658:Babuvirus / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification; D057166:High-Throughput Screening Assays; D028521:Musa / Q000821:virology*; D010935:Plant Diseases / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
chemical_list: D004279:DNA, Viral
keywords: Banana bunchy top virus; Quantitation; Real-time PCR; TaqMan(®) probe