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Safenkova I.V.,RAS A.N. Bach Institute of Biochemistry | Pankratova G.K.,RAS A.N. Bach Institute of Biochemistry | Zaitsev I.A.,Ag Lorch All Russian Potato Research Institute | Varitsev Y.A.,Ag Lorch All Russian Potato Research Institute | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2016

Multiarray on a test strip (MATS) was developed for the detection of eight important potato pathogens. The proposed assay combines the rapidity of immunochromatography with the high throughput of array techniques. The test zone of the immunochromatographic strip comprises ordered rows of spots containing antibodies specific for different potato pathogens. The assay benefits from the simplicity of immunochromatography; colored immune complexes form at the corresponding spots within the test zone. The presence and intensity of the coloration are used for identification of the target pathogens. The MATS was applied to the simultaneous detection of eight priority potato pathogens, characterized by the following limits of detection: 1 ng/mL for potato virus X and the ordinary type of potato virus Y, 10 ng/mL for potato virus M, 20 ng/mL for potato leaf roll virus, 40 ng/mL for necrotic-type potato virus Y, 100 ng/mL for potato virus S, 300 ng/mL for potato virus A, and 104 cells/mL for Clavibacter michiganensis subsp. sepedonicus. Analysis time was 15 min. The observed sensitivity of the MATS was comparable to the traditional enzyme-linked immunosorbent assay. The developed technique was tested on potato leaf extracts, and its efficiency for on-site control of the pathogens was confirmed in 100 % by commercial LFIA test strips. [Figure not available: see fulltext.] © 2016, Springer-Verlag Berlin Heidelberg.


Safenkova I.V.,RAS A.N. Bach Institute of Biochemistry | Zaitsev I.A.,Ag Lorch All Russian Potato Research Institute | Varitsev Yu.A.,Ag Lorch All Russian Potato Research Institute | Zherdev A.V.,RAS A.N. Bach Institute of Biochemistry | Dzantiev B.B.,RAS A.N. Bach Institute of Biochemistry
Biosciences Biotechnology Research Asia | Year: 2015

Potato blackleg and soft rot caused by Pectobacterium atrosepticum lead to significant yield losses. The early detection of P. atrosepticum is essential for healthy potato seed tubers. The aim of this study was to develop a method to rapidly detect P. atrosepticum based on the lateral flow immunoassay (LFIA) technique. Rabbit polyclonal antibodies specific to various strains of P. atrosepticum were obtained. Conjugates of these antibodies with gold nanoparticles averaging 20 nm in diameter were synthesized. Optimal concentrations of antibodies and conjugates deposited on membranes of the test strips were determined. The developed LFIA is suitable for analyzing potato tubers and leaves and has a visual detection limit of 2 × 105 cells/mL and a duration time of 10 min. Simple, rapid preparation of samples consists of homogenization in extracting buffer. No cross-reactivities with other potato pathogens, such as Pectobacterium carotovorum subsp. Carotovorum and Dickeya dianthicola, or saprophytes of healthy potato plants were detected. The assay was tested on 30 lots of potato tubers. The LFIA results were confirmed by ELISA (100% concurrence) and PCR (87.5% for positive samples and 95.5% for negative samples). Diagnosis of potato blackleg and soft rot by LFIA requires no equipment or training to perform, is cost effective and can be used in the field to monitor infection-causing P. atrosepticum.


Panferov V.G.,RAS A.N. Bach Institute of Biochemistry | Safenkova I.V.,RAS A.N. Bach Institute of Biochemistry | Varitsev Y.A.,Ag Lorch All Russian Potato Research Institute | Drenova N.V.,All Russian Plant Quarantine Center | And 3 more authors.
Talanta | Year: 2016

Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 2×102 CFU/mL; 20 min) in comparison with the common analysis (LOD 2×103 CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965. © 2016 Elsevier B.V. All rights reserved.


PubMed | RAS A.N. Bach Institute of Biochemistry, All Russian Plant Quarantine Center and Ag Lorch All Russian Potato Research Institute
Type: | Journal: Analytical and bioanalytical chemistry | Year: 2016

Early detection of potato infections is essential for effective disease management. The aim of this study was to develop a lateral flow immunoassay (LFIA) for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani. Polyclonal antibodies specific to different strains of Dickeya were obtained from rabbits after immunization with bacterial cells of D. dianthicola and D. solani. Enzyme-linked immunosorbent assay testing with use of a wide range of bacterial species showed that the polyclonal antibodies detect closely related strains of D. dianthicola and D. solani. Cross-reactivity with widespread pathogenic bacteria (nine species) and saprophytes of healthy potato plants was not detected. The LFIA based on the obtained antibodies and gold nanoparticles with average diameter of 20nm was developed. Under optimized conditions, the LFIA method enabled the analysis of potato extracts within 10min, with a visual limit of detection of 110


PubMed | RAS A.N. Bach Institute of Biochemistry, All Russian Plant Quarantine Center and Ag Lorch All Russian Potato Research Institute
Type: | Journal: Talanta | Year: 2016

Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 210(2) CFU/mL; 20 min) in comparison with the common analysis (LOD 210(3) CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965.

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