Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani
Graphical abstract
Introduction
Pectinolytic bacteria belonging to the family Pectobacteriaceae cause one of the most damaging plant diseases, known as bacterial soft rot [1]. Many reports have described significant yield losses for potato (Solanum tuberosum) and related economic worldwide damage caused by Pectobacteriaceae (see reviews [1,2]). In recent decades, the spread of phytopathogens has accelerated, leading to the most severe consequences [1]. It is associated with both climatic and environmental changes and adaptive transformations of the phytopathogens themselves [3,4]. Since 2004, Dickeya solani has proven to be more aggressive than other Dickeya spp. and Pectobacterium and extremely harmful for potato crops in Europe [5]. Therefore, controlling this pathogen is vital and strictly regulated by the European and Mediterranean Plant Protection Organization [6,7]. At the same time, detection of D. solani by symptoms (vascular discoloration, water-soaked surface spots, tissue decay, blackleg) is almost impossible because other bacteria (Pectobacterium and Dickeya spp.) also show the same symptoms [1,5,8,9]. To detect D. solani in the early stages of the disease, when no symptoms are visible, is a critical task. In addition, proposed approaches for detection should provide rapid diagnosis in nonlaboratory conditions.
To date, several approaches have been proposed for detecting D. solani. The most sensitive and applicable for strain discrimination are the well-known DNA amplification methods [10]. Three real-time polymerase chain reaction (PCR) assays have been developed for D. solani: the first, a species-specific TaqMan assay, was based on whole genome sequences and proposed by Pritchard et al. [11]; the second, by Kelly et al. [12], was based on the fusA gene; and the third, by van Vaerenbergh et al. [13], was based on flagellin sequences (fliC). Real-time PCR with a microreactor device demonstrated sensitivity at 1 ng DNA per ml of the sample [14]. However, these assays are time consuming and demand laboratory conditions with a thermocycler and highly qualified personnel.
Meanwhile, the most rapid and applicable method for field diagnostics is lateral flow assay (LFA) [15,16]. Commonly, LFA is based on specific recognition of antigens by antibodies and the formation of immune complexes, including a detectable label on the test strip. All interactions occur during a sample flow along the test strip and take no more than 10–15 min. Previously published LFAs allowed the detection of 105 colony-forming units (CFU) of Dickeya spp. per ml but did not discriminate D. solani from D. dianthicola [17].
Prospective approach for highly sensitive and rapid detection of D. solani is to combine a DNA amplification and LFA [18]. In particular, isothermal DNA amplification methods are often used for plant pathogen detection and applied in different variants with LFA [19,20]. The essence of the combination is isothermal amplification of target DNA using primers containing labels. Furthermore, the labeled amplicons form detectable complexes on the test strip. An assay based on loop-mediated isothermal amplification (LAMP) and an LFA for Candidatus Liberibacter detection reached a limit of detection (LOD) equal to 10 pg of the target DNA in a sample [21], whereas point-of-care diagnostic systems have also been developed for Leifsonia xyli subsp. xyli [22] and Pectobacterium carotovorum [23]. LAMP is performed at 65 °C, demands 6 primers, and produces a large number of inhomogeneous DNA products [24]. Another method of isothermal DNA amplification is recombinase polymerase amplification (RPA), which is more appropriate for LFA. The key features of RPA are the reaction proceeding for 15–20 min at a temperature range of 30–42 °C, 2 primers, and 1 DNA product of predictable length [[25], [26], [27]]. Ahmed et al. developed a test based on RPA with the following LFA for a specific detection of Pectobacterium spp. in infected potato tubers [28]. The detection limit of the test was 10 fg of target DNA in a sample. The test could distinguish Pectobacterium from Dickeya and other pathogen species. RPA with LFA was developed for another bacterial phytopathogen's detection—Candidatus Liberibacter asiaticus [29]. Agdia, Inc. (Elkhart, USA) has commercialized rapid isothermal DNA amplification kits based on RPA for the detection of Clavibacter michiganensis subsp. sepedonicus (detection limit equal to 22 copies of its recombinant plasmid DNA/500 fg genomic DNA) and Dickeya spp. (detection limit ~2 × 102 CFU mL−1). Therefore, the RPA-LFA has shown efficiency for the detection of bacterial phytopathogens but has not yet been applied for the detection of D. solani.
In this study, an RPA-LFA test system was developed for specific detection of D. solani in potato tubers. The research included (1) design and screening of primers for PCR and RPA; (2) synthesis and characterization of the LFA components; (3) an LOD determination for RPA-LFA with the chosen primers; (4) checking of the cross-reactivity with other species of Dickeya, Pectobacterium, and nonrelated potato pathogens; and (5) detection of D. solani in tuber extracts using RPA-LFA and verification of the obtained results through PCR.
Section snippets
Preparation of bacterial samples
D. solani (DSM 28711), D. dianthicola (DSM 18054), D. zea (DSM 18068), D. dadantii subsp. dadantii (DSM 18020), D. paradisiaca (DSM 18069), D. fangzhongdai (DSM 101947), D. chrysanthemi (DSM 4610), P. atrosepticum (DSM 18077), and P. carotovorum subsp. carotovorum (DSM 30168) were obtained from the collection of phytopathogens of the Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures (Germany). C. michiganensis subsp. sepedonicus (Rf355) was acquired from the
Primers design
First, specific primers were designed to detect D. solani. Four regions in the genome of D. solani were chosen as possible targets for the detection, which were previously described as specific regions for D. solani: recA gene [31], IGS [32], the SOL-A region (fragment of FlgH gene), and the SOL-C region (nonannotated gene) [11]. Based on these genome regions, two types of primers (SOL-A, SOL-C [11]) were proposed for the PCR detection of D. solani; the SOL-C fragment has low coverage with D.
Conclusions
In this study, a rapid (20 min duration of RPA and 10 min duration of LFA) and highly sensitive RPA-LFA for detection of D. solani was developed. After screening of primers chosen from 4 possible target-specific genomic regions, SOL-C region was selected as the target sequence for detection. The combination of isothermal RPA with an LFA of the product of the reaction showed sensitivity of 14,000 CFU of D. solani per gram of potato tuber (700 CFU per mL of the tested extract sample). This is
Funding
This study was financially supported by the Russian Science Foundation (Grant 16-16-04108).
CRediT authorship contribution statement
Aleksandr V. Ivanov: Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Irina V. Safenkova: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing - original draft, Writing - review & editing, Funding acquisition. Natalia V. Drenova: Investigation, Resources, Writing - review & editing. Anatoly V. Zherdev: Conceptualization, Methodology, Visualization, Supervision, Writing -
Declaration of competing interest
The authors declare no conflict of interest.
References (37)
- et al.
Virulence program of a bacterial plant pathogen: the Dickeya model
Prog. Mol. Biol. Transl.
(2016) Infectious pathogens meet point-of-care diagnostics
Biosens. Bioelectron.
(2018)- et al.
Signal amplification strategies for paper-based analytical devices
Biosens. Bioelectron.
(2019) - et al.
Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics
Biosens. Bioelectron.
(2015) - et al.
Recombinase polymerase amplification: basics, applications and recent advances
Trac. Trends Anal. Chem.
(2018) - et al.
Nucleic acid lateral flow assay with recombinase polymerase amplification: solutions for highly sensitive detection of RNA virus
Talanta
(2020) - et al.
Key significance of DNA-target size in lateral flow assay coupled with recombinase polymerase amplification
Anal. Chim. Acta
(2020) The changing face of bacterial soft-rot diseases
Annu. Rev. Phytopathol.
(2018)- et al.
Top 10 plant pathogenic bacteria in molecular plant pathology
Mol. Plant Pathol.
(2012) Migrate or evolve: options for plant pathogens under climate change
Global Change Biol.
(2013)
Dickeya species: an emerging problem for potato production in Europe
Plant Pathol.
Diagnostic Protocols for Regulated Pests, EPPO Standard PM 7, EPPO Standards: General Phytosanitary Measures
EPPO A1 and A2 Lists of Pests Recommended for Regulation as Quarantine Pests, EPPO Standard PM 1/2, EPPO Standards: General Phytosanitary Measures
Dickeya undicola sp. nov., a novel species for pectinolytic isolates from surface waters in Europe and Asia
Int. J. Syst. Evol. Microbiol.
Compendium of Potato Diseases
Sensitive molecular diagnostic assays to mitigate the risks of asymptomatic bacterial diseases of plants
Crit. Rev. Immunol.
Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences
Plant Pathol.
Development of a real-time PCR assay for the detection of ‘Dickeya solani’
Proc. Crop Protect. Northern Br.
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