Abstract
Pectobacterium species cause serious bacterial soft rot diseases worldwide on economically important fruit and vegetable crops including tomato and potato. Accurate and simple methods are essential for rapid pathogen identification and timely management of the diseases. Recombinase polymerase amplification (RPA) combined with a lateral flow device (LFD) was developed for specific detection of Pectobacterium sp. directly from infected plant materials with no need for DNA isolation. The specificity of RPA-LFD was tested with 26 Pectobacterium sp. strains and 12 non-Pectobacterium species and no false positive or false negative outcomes were observed. RPA primers and probe for host control were also developed to detect the host genome for enhanced reliability and accuracy of the developed assay. The detection limit of 10 fg was obtained with both sensitivity and spiked sensitivity assays. No inhibitory effects were observed on the RPA assay when targets (pathogen and host) were directly detected from infected potato and tomato sap. The developed RPA assay has numerous applications from routine diagnostics at point-of-care, biosecurity, surveillance and disease management to epidemiological studies. In addition, this tool can also be used to discover reservoir hosts for Pectobacterium species.
Highlights
Soft rot bacteria in the genus Pectobacterium affect a wide range of host plants worldwide and cause significant economic losses in the field, storage and during transit[1]
The ring image output shows a comparison of a reference genome sequence of P. carotovorum colored with dark blue in the center circle with other closely and distantly related bacterial genomes, P. carotovorum subsp. carotovorum (NC_18525), P. carotovorum subsp. brasiliensis (NZ_CP020350) P. atrosepticum (NZ_CP009125), P. wasabiae (NC_013421), D. zeae (NZ_CP006929), D. dadantii (NC_014500), D. solani (NZ_CP009460), E. amylovora (NC_013961), Figure 2
We have established a novel lateral flow strip-based recombinase polymerase amplification (RPA) assay for Pectobacterium detection that provides unique advantages with respect to speed, specificity, sensitivity, ease of visual detection and simplicity of equipment required
Summary
Soft rot bacteria in the genus Pectobacterium (formerly Erwinia in the family Enterobacteriaceae) affect a wide range of host plants worldwide and cause significant economic losses in the field, storage and during transit[1]. PCR-based methods have some disadvantages - they are time-consuming, require a sophisticated and expensive thermal cycler, and cannot conveniently be used at point-of-care[8]. Can be performed at point-of-care without the need of an expensive thermal cycler 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]. RPA has a comparatively low temperature requirement (37 °C to 42 °C) and freeze-dried TwistDx RPA reagents are commercially available This is a highly sensitive, rapid and accurate isothermal detection method that can be performed at the point-of-care with minimum requirements[17]. The assay has applications in routine diagnostic surveys for biosecurity, disease management and disease epidemiology
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