Establishment of a loop-mediated isothermal amplification (LAMP) assay for the detection of phytoplasma-associated cassava witches’ broom disease

详细信息    查看全文

• 作者：Nam Tuan Vu ; Juan Manuel Pardo ; Elizabeth Alvarez…
• 刊名：Journal of the Korean Society for Applied Biological Chemistry
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：59
• 期：2
• 页码：151-156
• 全文大小：1,756 KB
• 刊物主题：Applied Microbiology; Bioorganic Chemistry; Biological Techniques;
• 出版者：Springer Netherlands
• ISSN：2234-344X
• 卷排序：59

文摘

Cassava (Manihot esculenta Crantz) is one of the most important food crops in the tropics; however, bacterial phytopathogens pose a serious threat to its farming. Cassava Witches’ Broom Disease (CWB) is caused by the infection of phytoplasma and is manifested as reduction in tuber yield and starch content at harvest of 10 and 30 %, respectively. Although polymerase-chain reaction provides the gold standard in diagnostics, this method requires significant investments in infrastructure and training. Here, we developed a loop-mediated isothermal amplification (LAMP) assay that allows specific detection of phytoplasma from field-collected samples. Three primer sets were designed, of which two detected phytoplasma DNA sequence encoding 16S rRNA (16S rDNA), the other detected cassava actin. Following a 1 h LAMP reaction at 63 °C, a positive reaction can be visualized by agarose gel electrophoresis, hydroxynaphthol blue color change, or the presence of a precipitate. In a pilot field study, the assay was able to rapidly distinguish between healthy and CWB-infected cassava. With further development, a LAMP for routine on-site screening of cassava crops can be envisioned.KeywordsCassavaCassava witches’ broom diseaseLoop-mediated amplificationLoop-mediated isothermal amplificationPhytoplasma