Chiquito-Almanza Elizabeth, Acosta-Gallegos Jorge A, García-Álvarez Nadia C, Garrido-Ramírez Eduardo R, Montero-Tavera Victor, Guevara-Olvera Lorenzo, Anaya-López José L
Instituto Tecnológico de Celaya, Departamento de Ingeniería Bioquímica, Celaya, Guanajuato, CP 38010, Mexico.
INIFAP-Campo Experimental Bajío, Celaya, Guanajuato, CP 38010, Mexico.
Viruses. 2017 Mar 30;9(4):63. doi: 10.3390/v9040063.
A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed to simultaneously detect bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV), and bean golden yellow mosaic virus (BGYMV) from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB) method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the gene of BGYMV as well as 469 bp and 746 bp from the gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%), 41/187 (21.9%), and 35/187 (18.7%), for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5%) samples were co-infected with two of these viruses and 10/187 (5.3%) with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.
开发了一种多重逆转录聚合酶链反应（RT-PCR）检测方法，使用单一的十六烷基三甲基溴化铵（CTAB）总核酸提取方法，从用硅胶干燥的普通菜豆叶片中同时检测菜豆普通花叶病毒（BCMV）、菜豆普通花叶坏死病毒（BCMNV）和菜豆金黄花叶病毒（BGYMV）。使用五种特异性引物的混合物扩增三个不同的片段，分别对应于BGYMV基因的272 bp以及BCMV和BCMNV基因的469 bp和746 bp。这三种病毒可在单株植物或五株植物的混合样本中检测到。多重RT-PCR成功应用于从墨西哥瓜纳华托州、纳亚里特州和哈利斯科州23个市收集的187份田间样本中检测这三种病毒。BGYMV、BCMV和BCMNV的单感染率分别为14/187（7.5%）、41/187（21.9%）和35/187（18.7%）；29/187（15.5%）的样本被其中两种病毒共同感染，10/187（5.3%）的样本被三种病毒共同感染。这种多重RT-PCR检测方法是一种简单、快速、灵敏且经济高效的检测普通菜豆中这些病毒的方法，可用于常规分子诊断和流行病学研究。
