Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, United Kingdom.
J Virol Methods. 2012 Jan;179(1):176-84. doi: 10.1016/j.jviromet.2011.10.020. Epub 2011 Nov 3.
Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocols were developed for the detection of cassava brown streak viruses (CBSVs) in single and mixed infections with cassava mosaic begomoviruses (CMBs) in a tropical crop plant, cassava (Manihot esculenta). CMBs contain ssDNA as their genome (genus Begomovirus, family Geminiviridae) while CBSVs are made up of positive sense ssRNA (genus Ipomovirus, family Potyviridae), and they cause the economically important cassava mosaic and cassava brown streak diseases, respectively, in sub-Saharan Africa. Diagnostic methodologies have long been available for CMBs but they are limited for CBSVs especially in mixed infections. In this study, the two CBSVs, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) occurring singly or in mixed infection with CMBs, African cassava mosaic virus and East African cassava mosaic virus were detected in a single RT-PCR using both previously described and newly designed virus-specific primers. These protocols were highly efficient for detecting CBSVs compared to the existing methods and have great potential to minimize sample handling and contamination. As well as improving the diagnosis of cassava viruses, the development of multiplex RT-PCR protocols have revealed the common occurrence of mixed infections by CBSV and CBSUV in cassava fields of Tanzania and Kenya, which was contrary to the common belief until recently that these two viruses have existed separately. These protocols have implications for diagnosis and epidemiological studies on cassava virus diseases in Eastern Africa.
建立了用于检测热带作物木薯中单一和混合感染木薯曲叶病毒（CMB）的单孢和多孢逆转录-聚合酶链反应（RT-PCR）检测方法。CMB 含有 ssDNA 作为其基因组（单属病毒，双生病毒科），而 CBSV 由正单链 RNA 组成（属 Ipomovirus，马铃薯 Y 病毒科），它们分别导致经济上重要的木薯花叶病和木薯褐色条斑病在撒哈拉以南非洲。长期以来，一直有针对 CMB 的诊断方法，但针对 CBSV 的方法有限，特别是在混合感染的情况下。在这项研究中，两种 CBSV，即木薯褐色条斑病毒（CBSV）和乌干达木薯褐色条斑病毒（CBSUV），在单独或与 CMB、非洲木薯花叶病毒和东非木薯花叶病毒混合感染的情况下，使用两种先前描述和新设计的病毒特异性引物，在单个 RT-PCR 中进行了检测。与现有方法相比，这些方案在检测 CBSV 方面非常有效，并且具有很大的潜力可以最小化样品处理和污染。除了改进木薯病毒的诊断外，多 RT-PCR 方案的开发还揭示了在坦桑尼亚和肯尼亚的木薯田中 CBSV 和 CBSUV 混合感染的常见发生，这与直到最近的普遍看法相反，即这两种病毒一直是单独存在的。这些方案对东非木薯病毒病的诊断和流行病学研究具有重要意义。
