Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses.

Abstract:

:The genus Badnavirus is characterized by members that are genetically and serologically heterogeneous which presents challenges for their detection and characterization. The presence of integrated badnavirus-like sequences in some host species further complicates detection using PCR-based protocols. To address these challenges, we have assessed and optimized various RCA protocols including random-primed RCA (RP-RCA), primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-RCA) and specific-primed RCA (SP-RCA). Using Dioscorea bacilliform AL virus (DBALV) as an example, we demonstrate that viral DNA amplified using the optimized D-RCA and SP-RCA protocols showed an 85-fold increase in badnavirus NGS reads compared with RP-RCA. The optimized RCA techniques described here were used to detect a range of badnaviruses infecting banana, sugar cane, taro and yam demonstrating the utility of RCA for detection of diverse badnaviruses infecting a variety of host plant species.

journal_name

Virology

journal_title

Virology

authors

Sukal AC,Kidanemariam DB,Dale JL,Harding RM,James AP

doi

10.1016/j.virol.2019.01.013

subject

Has Abstract

pub_date

2019-03-01 00:00:00

pages

73-80

eissn

0042-6822

issn

1096-0341

pii

S0042-6822(19)30013-3

journal_volume

529

pub_type

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