Duplex recombinase polymerase amplification assay for simultaneous detection of Pythium spp. and Ralstonia pseudosolanacearum from rhizomes
Duplex recombinase polymerase amplification assay for simultaneous detection of Pythium spp. and Ralstonia pseudosolanacearum from rhizomes
复制标题
DOI：
10.1016/j.cropro.2022.106057
复制
发表时间：
2022-07-21
期刊：
影响因子：
2.8
通讯作者：
Punya, K. C.
中科院分区：
文献类型：
--
作者：
Jeevalatha, A.;Zumaila, Fathimath;Punya, K. C.
软腐病和青枯病是世界范围内限制生姜生产的主要病害。由于这些病原菌主要通过被广泛用作种植材料的生姜根茎传播，因此需要有效和灵敏的技术来进行可靠和准确的诊断。在本研究中，建立了单链和双链重组酶聚合酶扩增(RPA)方法，用于腐霉的特异性和敏感性检测。和从生姜根茎中分离到的拟青枯雷尔氏菌(Ralstia Passosolanacearum)。双重RPA方法对拟青枯病菌和腐霉的检测灵敏度分别为10倍和100倍，且与其他生姜根茎病原菌如镰刀菌、菜豆巨藻和菌核无交叉扩增，具有较高的特异性。此外，这些分析可以在加热块中37至40摄氏度的等温条件下进行。在验证试验中，从田间、储藏和市场上采集的生姜根茎样品中提取的粗DNA可以成功地检测到这些病原菌。此外，建立的RPA方法可以检测出聚合酶链式反应阴性的样品中的病原体。本研究可能是首次用双重RPA方法同时检测生姜中的真菌和细菌病原体。
Soft rot and bacterial wilt are the major production constraints in ginger worldwide. As these pathogens are transmitted primarily through ginger rhizomes that are widely used as planting materials, effective and sensitive techniques are required for reliable and accurate diagnosis. In the present study, uniplex and duplex recombinase polymerase amplification (RPA) assays were developed for specific and sensitive detection of Pythium spp. and Ralstonia pseudosolanacearum from ginger rhizomes. The duplex RPA assay was 10 and 100 times more sensitive than duplex PCR assay in case of R. pseudosolanacearum and Pythium spp., respectively and were highly specific as they did not show any cross amplification with other rhizome-borne pathogens of ginger such as Fusarium spp., Macrophomina phaseolina and Sclerotium rolfsii. In addition, the assays could be performed under isothermal conditions at a temperature ranging from 37 to 40 degrees C in a heating block. In validation tests, these pathogens could be successfully detected using crude DNA extracted from ginger rhizome samples collected from the field, storage and market. In addition, the RPA assay developed could detect the pathogens in samples which were found negative in PCR assay. The present study could be the first report of simultaneous detection of fungal and bacterial pathogens using duplex RPA assay in ginger.