Centre for Tropical Crops and Biocommodities (CTCB), Faculty of Science and Engineering (SEF), Queensland University of Technology (QUT), Brisbane 4001, Australia; Centre for Pacific Crops and Trees (CePaCT), Land Resource Division (LRD), Pacific Community (SPC), Suva, Fiji.
Centre for Tropical Crops and Biocommodities (CTCB), Faculty of Science and Engineering (SEF), Queensland University of Technology (QUT), Brisbane 4001, Australia.
Virology. 2019 Mar;529:73-80. doi: 10.1016/j.virol.2019.01.013. Epub 2019 Jan 15.
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.
该属 Badnavirus 的特征是成员在遗传和血清学上存在异质性，这给它们的检测和特征描述带来了挑战。一些宿主物种中存在整合的类似 badnavirus 序列，这进一步增加了使用基于 PCR 的方案进行检测的复杂性。为了解决这些挑战，我们评估和优化了各种 RCA 方案，包括随机引物 RCA（RP-RCA）、引物添加的随机引物 RCA（primer-spiked RP-RCA）、定向 RCA（D-RCA）和特异性引物 RCA（SP-RCA）。以 Dioscorea bacilliform AL 病毒（DBALV）为例，我们证明，使用优化的 D-RCA 和 SP-RCA 方案扩增的病毒 DNA，与 RP-RCA 相比，badnavirus NGS 读长增加了 85 倍。本文描述的优化 RCA 技术用于检测感染香蕉、甘蔗、芋头和山药的一系列 badnaviruses，证明了 RCA 技术在检测感染各种宿主植物的各种 badnaviruses 方面的实用性。
