Specific detection of Pectobacterium carotovorum by loop‐mediated isothermal amplification

Abstract

Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.

Genome-informed diagnostics for specific and rapid detection of Pectobacterium species using recombinase polymerase amplification coupled with a lateral flow device

“…LAMP methods for detection of the closely related genera Pectobacterium and Dickeya have already been developed by Yasuhara-Bell and coworkers 16 , but LAMP assays have the disadvantage of requiring a high temperature for amplification (65 °C) and an expensive instrument. RPA is a comparatively new isothermal technique that can detect target DNA in 15–30 min at 37–42 °C with no requirement for sophisticated and/or expensive instruments 20 .…”

“…These include: strand displacement amplification (SDA) 10 , loop-mediated isothermal amplification (LAMP) 11 , rolling circle amplification (RCA) 12 , helicase-dependent amplification (HDA) 13 , recombinase polymerase amplification (RPA) 14 and nicking enzyme amplification reaction (NEAR) 15 . Yasuhara‐Bell and coworkers 16 (2016) reported the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect the blackleg pathogen, P . atrosepticum and soft rot pathogen, P .…”

A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac

“…Previous studies have reported on the LAMP-based detection of P. carotovorum [28]. However, detection technology regarding P. aroidearum has not been previously reported.…”

“…To date, LAMP assays have been applied to the diagnosis of some Pectobacterium species [28]. However, these assays are not capable of specifically detecting P. aroidearum .…”

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

“…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 .…”