Cloning of BBTV (Banana Bunchy Top Virus) components and screening of BBTV using functionalized gold nanoparticles

Banana bunchy top virus (BBTV) affects all varieties of banana plants and causes heavy economic loss in most of the banana cultivating areas. The BBTV genome comprises of six DNA components; in this study, we have cloned the six BBTV-DNA components from one of the BBTV-infected plants (Tri-8) and were submitted to GenBank. Analysis of the BBTV DNA-R component showed that it belonged to south Pacific group. Resistance against BBTV has not been observed so far in banana plants and removal and killing of the infected plants has been routinely practiced. Hence, early detection of BBTV infection would be desirable and various detection methods routinely employed include enzyme linked immunosorbent assay (antigen-antibody based) and molecular-based methods such as polymerase chain reaction (PCR), qPCR, or LAMP PCR. Most of these methods require enzymes or antibodies for detection and hence are expensive. Here, we report a visual detection method (AuNP probe assay) using gold nanoparticles (AuNPs) functionalized with an ssDNA-thiolated probe (CR1). This method is based on the hybridization of the functionalized AuNPs with the target DNA (BBTV). In the AuNP probe assay, the functionalized AuNPs retains red colour when BBTV DNA is present, and in the absence of BBTV DNA, the colour of the functionalized AuNPs changes to purple when salt is added. The AuNP probe assay was compared with PCR for the detection of banana plants and it was found that AuNP probe assay was better than PCR in detecting BBTV infection (86.5% for AuNP probe assay and 65% for PCR). The AuNP probe assay was found to be highly specific to BBTV and was found to detect up to 1 pg/μl of the plasmid (pTZBBTri 4, BBTV DNA) mixed with healthy banana DNA.