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

Abstract

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.

Detection of Begomovirus in Chilli and Tomato Plants Using Functionalized Gold Nanoparticles Authors: Lavanya. R and Arun. V

“…In our lab, we have developed an AuNP probe-based method for the detection of BBTV in banana plants and was found to be better than PCR (53). Overall, the detection e ciency of AuNP assay (77.7%) was found to be better than PCR screening (49.4%) in case of chilli plants.…”

“…Recently, a visual DNA method using integrated Recombinase Polymerase ampli cation (RPA) and a AuNP probe has been reported for detection of tomato yellow leaf curl virus and was found to be highly sensitive and stable (52). In our lab, we had developed a method for detection of Banana Bunchy Top Virus (BBTV) using functionalized AuNPs (53). In this work, we demonstrated the applicability of functionalized AuNPs in the detection of begomoviruses in plants and an Indian patent has been led (Application No: 201941007582).…”

Detection of Begomovirus in chilli and tomato plants using functionalized gold nanoparticles

“…Functionalization of thiolated probes. The thiolated probes (10 nM) were functionalized by treating with freshly prepared dithiothreitol (DTT) and purified as described in Kumar et al 53 . Briefly, 1 ml of 10 nM thiolated probe was treated with 100 µl of DTT and incubated for 30 min at RT and purified using NAP25 column as per manufacturer's instructions (GE Lifesciences) and used for conjugation of gold nanoparticles (AuNPs).…”

Genetic Structure and Diversity of Banana Bunchy Top Virus (BBTV) in the Philippines

“…The disease transmission is of the persistent, circulative, non-propagative type (Anhalt and Almeida, 2008), with efficiency ranging from 46-67% (Magee, 1927; Wu and Su., 1990; Hu et al, 1996). The virus is made up of 6 genetic components, namely, DNA-C coding for the cell cycle link protein, DNA-S coding for the capsid protein, DNA-M coding for the movement protein, DNA-N coding for the nuclear shuttle protein, DNA-U3 coding for potential protein with unknown function, and DNA-R coding for the replication initiation protein (Amin et al, 2008, Kumar et al, 2017; Wickramaarachchi et al, 2016).…”