Rolling Circle Amplification Is More Sensitive than PCR and Serology-Based Methods in Detection of <i>Banana streak virus</i> in <i>Musa</i> Germplasm

Abstract

Banana (Musa sp.) is a popular and important crop among many communities in East Africa. Banana production is however threatened by the wide-spread banana streak disease (BSD), caused by Banana streak virus (BSV). The success of BSV management is inherently coupled to the availability of a sensitive indexing method. In this study, the sensitivity of three BSV detection techniques: rolling circle amplification (RCA), immunocapture PCR (with degenerate and Gold finger primers) and standard PCR was compared. A set of 32 BSD-asymptomatic samples were used to compare the techniques. Analysis of variance (ANOVA) for comparison of the four techniques showed that there were significant differences (P < 0.05) among all the means, with RCA and direct PCR having the highest detection mean values. Owing to its fidelity and capacity to circumvent the amplification of the integrated nuclear viral sequences, the RCA technique is recommended for routine indexing of Musa tissues for BSV. This study unveils a more reliable BSV detection method, a need that has remained unaddressed for a long while.

Episomal detection of Banana streak OL virus in single and mixed infection with Cucumber mosaic virus in banana 'Nanicão Jangada'

“…Results of this study confirmed previous works (James et al, 2011;Wambulwa et al, 2012) that RCA is very reliable to detect episomal BSV, as the sequenceindependent nature of RCA can selectively amplify circular and not linear (integrated) DNA. RCA specifically amplified the genome of episomal BSV present in 'Nanicão Jangada' (Table 1, Figure 2).…”

Badnaviruses: The Current Global Scenario

“…RCA would also aid in the detection and identification of new badnaviruses. The RCA combined with RFLP was successfully employed in the detection of BSV species and FBV-1 [ 55 , 73 , 77 ]. A combination of immunosorbent electron microscopy (ISEM), IC-PCR, RCA, virus purification, Southern and in situ hybridizations, and complete sequencing of the virus genome may be required to elucidate the precise identification of the type of sequences present.…”

“…Multiplex immunocapture PCR (M-IC-PCR) and rolling circle amplification (RCA) have been developed for the detection of BSV genomes [ 72 , 73 , 104 ]. RCA was more sensitive than PCR and serology-based methods in the detection of BSV [ 77 ]. Selvarajan et al [ 62 ] developed serological-based detection of BSMYV using the recombinant virus-associated protein encoded by ORF II, which is very specific to each species of BSV.…”

A Sequence-Independent Strategy for Amplification and Characterisation of Episomal Badnavirus Sequences Reveals Three Previously Uncharacterised Yam Badnaviruses

“…RCA is a sequence-independent approach that is often used in the laboratory for the amplification of circular DNA virus genomes, thus overcoming the shortcoming of primer dependency in PCR for the amplification of viruses with a circular DNA genome [ 33 , 38 , 39 ]. The RCA technique has been used successfully for the study of several other circular DNA viruses such as sweet potato leaf curl virus [ 40 ] and beet curly top virus [ 41 ] of the family Geminiviridae , and BSV [ 39 , 42 ] and fig badnavirus 1 [ 34 ] of the family Caulimoviridae. It was proposed that RCA could be used for the specific detection of episomal forms of yam badnaviruses [ 30 ].…”