Development of a Loop-Mediated Isothermal Amplification Assay for the Detection of Dickeya spp.

Abstract

Dickeya and Pectobacterium spp. are responsible for soft-rotting diseases of several plant species, some with overlapping host range. On potato, symptoms caused by these pathogens cannot be clearly differentiated. Disease results in the downgrading and rejection of potato seed, thus requiring additional phytosanitary restrictions across Northern Europe and other parts of the world. In an effort to provide a more timely and accurate diagnostic to distinguish these two groups of pathogens, a method for detecting Dickeya spp. using loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay can be used to test crude extracts prepared directly from symptomatic lesions. The entire test can be completed in less than 30 min, making it faster than the current diagnostic standard, the pelADE conventional polymerase chain reaction. Additionally, the LAMP assay was able to detect Dickeya DNA in samples spiked with varying amounts of Pectobacterium DNA, thus demonstrating the highly specific and sensitive nature of the assay, which can be applied on survey samples with mixed soft-rotting bacterial populations.

Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real‐time qPCR assay to detect the genus Dickeya and Dickeya dianthicola

“…Detection based on molecular methods such as PCR, quantitative PCR and loop‐mediated isothermal amplification (LAMP) are implemented routinely due to their high sensitivity, speed, specificity, accuracy, discriminatory ability and reproducibility (Ouyang et al ; Czajkowski et al ; Larrea‐Sarmiento et al ; Ocenar et al ). Yasuhara‐Bell et al () also developed a LAMP assay for detection of Dickeya sp. using mglC gene region.…”

Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria

“…Compared to conventional nucleic acid-based methods, LAMP is rapid and avoids the need of sophisticated laboratory equipment like PCR and qPCR machines 25 . With the forward and backward loop primers, results can be obtained even in less than 20 minutes.…”

“…With the forward and backward loop primers, results can be obtained even in less than 20 minutes. There are numerous chemistries and several instruments used for LAMP detection from colorimetric, lateral flow device, portable battery-operated instruments to qPCR 25 , 32 . We used a field deployable battery operated small (D = 8 cm × W = 14 cm × H = 7 cm) Bioranger TM instrument for the real-time detection of reaction amplification that makes the assay easy to use for field applications.…”

“…Most importantly, it can be easily performed at the point-of-care. It has been successfully used for rapid and specific detection of plant bacteria from infected plant tissues and soil 24 , 25 .…”

Detection of Potato Virus Y (Pvy) by Reverse-Transcription Loop-Mediated Nucleic Acid Amplification (Rt-Lamp)

“…During the last decade several variants of RT-LAMP have been developed to detect PVY (Nie, 2005;Almasi and Dehabadi, 2013;Hasiów-Jaroszewska et al, 2015;Przewodowska et al, 2015, Treder et al, 2018 as well as other plant viruses (Varga and James, 2006;Ahmadi et al, 2013;Tomlinson et al, 2013;Hasiów-Jaroszewska and Borodynko, 2013;Shen et al, 2014;Budziszewska et al, 2016) and Potato spindle tuber viroid (Lenarčič et al, 2012). The LAMP protocols have also recently been published for Phytophtora infestans (Hansen et al, 2016;Khan et al, 2017;Si Ammour et al, 2017), Pectobacterium carotovorum (Yasuhara-Bell et al, 2016) Pectobacterium atrosepticum (Hu et al, 2016), Dickeya spp (Yasuhara-Bell et al, 2017) and Ralstonia solanacearum (Lenarčič et al, 2014).…”