vestichИзвестия Национальной академии наук Беларуси. Серия химических наукProceedings of the National Academy of Sciences of Belarus, Chemical Series1561-83312524-2342The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1561-8331-2024-60-4-314-325vestich-917Research ArticleБИООРГАНИЧЕСКАЯ ХИМИЯBIOORGANIC CHEMISTRYКомбинированные системы полимеразной цепной реакции и иммуноанализа с времяразрешенной флуориметрией или мембранной иммунохроматографией для количественного определения ДНК бактерий Salmonella entericaCombined systems of polymerase chain reaction and a time-resolved fluorescence immunoassay or membrane immunochromatography for quantitative determination of Salmonella enterica bacterial DNAСерченяТ. С.SerchenyaT. S.

Серченя Татьяна Сергеевна – кандидат химических наук, доцент, ведущий научный сотрудник

ул. Академика В. Ф. Купревича, 5/2, 220084, Минск

Serchenya Tatyana S. – Ph. D. (Chemistry), Associate Professor, Leading Researcher

5/2, Academician V. F. Kuprevich Str., 220084, Minsk

serchenya@iboch.byОхремчукЕ. В.AkhremchukK. U.

Охремчук Екатерина Владимировна – кандидат биологических наук, научный сотрудник

ул. Академика В. Ф. Купревича, 2, 220084

Akhremchuk Katsiaryna U. – Ph. D. (Biology), Researcher

2, Academician V. F. Kuprevich Str., 220084, Minsk

katerina_akhr@bio.bsu.byВалентовичЛ. Н.ValentovichL. N.

Валентович Леонид Николаевич – кандидат биологических наук, доцент, заведующий лабораторией

ул. Академика В. Ф. Купревича, 2, 220084

Valentovich Leonid N. – Ph. D. (Biology), Head of Laboratory

2, Academician V. F. Kuprevich Str., 220084, Minsk

valentovich@mbio.bas-net.byЛапинаВ. С.LapinaV. S.

Лапина Виктория Сергеевна – младший научный сотруд- ник

ул. Академика В. Ф. Купревича, 5/2, 220084, Минск

Lapina Victoryia S. – Junior Researcher

5/2, Academician V. F. Kuprevich Str., 220084, Minsk

lapina@iboch.byСвиридовО. В.SviridovO. V.

Свиридов Олег Васильевич – доктор химических наук, про- фессор, заведующий лабораторией

ул. Академика В. Ф. Купревича, 5/2, 220084, Минск

Sviridov Oleg V. – D. Sc. (Chemistry), Professor, Head of Laboratory

5/2, Academician V. F. Kuprevich Str., 220084, Minsk

sviridov@iboch.byИнститут биоорганической химии Национальной академии наук БеларусиInstitute of Bioorganic Chemistry of the National Academy of Sciences of BelarusИнститут микробиологии Национальной академии наук БеларусиInstitute of Microbiology of the National Academy of Sciences of Belarus202430112024604314325Copyright © Серченя Т.С., Охремчук Е.В., Валентович Л.Н., Лапина В.С., Свиридов О.В., 20242024Серченя Т.С., Охремчук Е.В., Валентович Л.Н., Лапина В.С., Свиридов О.В.Serchenya T.S., Akhremchuk K.U., Valentovich L.N., Lapina V.S., Sviridov O.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestichem.belnauka.by/jour/article/view/917

Разработаны и исследованы четыре модельные биоаналитические системы, специфичные к бактериям Salmonella enterica, в которых в результате полимеразной цепной реакции (ПЦР) продуцировался содержащий остатки биотина и флуоресцеина ампликон ДНК. Это позволило иммобилизовать ампликон в функционализиро- ванной твердой фазе и биоспецифически пометить хелатом европия в микропланшетах или золотыми наночастицами на хроматографической мембране. Количественная детекция модифицированной ДНК осуществлялась в системах иммуноанализа с времяразрешенной флуоресценцией Eu3+ (лантанидный иммунофлуоресцентный анализ, ЛИФМА, DELFIA) или с фотометрией окрашенной зоны на хроматографической полоске (ИХА). Разработаны и исследованы три пары праймеров для получения выбранных фрагментов гена invA, присутствующего в геномах всех патогенных сальмонелл. Установлена их пригодность для тест-систем. В микропланшетной системе ЛИФМА диапазон определяемых концентраций полученного ампликона ДНК составил 0,01–10,0 нМ, а предел обнаружения оказался равным 2 пМ. Предел визуальной детекции ампликонов ДНК в ИХА составил 0,05 нМ. Показана возможность проведения тестирования ампликонов без дополнительного выделения ДНК в чистом виде из реакционной смеси. Установлена высокая специфичность разработанных биоаналитических систем для детекции Salmonella enterica различных серотипов.

Four model bioanalytical systems specific for Salmonella enterica have been developed and studied, in which a polymerase chain reaction (PCR) produced a DNA amplicon containing biotin and fluorescein residues. This enabled to immobilize the amplicon on a functionalized solid phase and to label it biospecifically with europium chelate in microplates or gold nanoparticles on a chromatographic membrane. Quantitative detection of the modified DNA was carried out in immunoassay systems by measuring the Eu3+ time-resolved fluorescence (dissociation-enhanced lanthanide fluorescence immunoassay, DELFIA) or by photometry of the colored zone on the chromatographic strip (LFA). Three pairs of primers were developed and examined to obtain selected fragments of the invA gene, which is present in the genomes of all pathogenic Salmonella enterica. The fragments proved to be suitable for the test systems. In the microplate DELFIA system, the concentration range of DNA amplicon quantification was found to be 0.01–10.0 nM, and a detection limit was 2 pM. The limit of DNA visual detection in LFA was 0.05 nM. The possibility of testing the amplicons without additional isolation of pure DNA from the reaction mixture was demonstrated. The high specificity of the developed bioanalytical systems for the detection of various Salmonella enterica serotypes was demonstrated.

патогенные бактерииSalmonella entericaполимеразная цепная реакцияиммуноанализ с времяразрешенной флуориметриейиммунохроматографический анализpathogenic bacteriaSalmonella entericapolymerase chain reactiondissociation-enhanced lanthanide fluorescence immunoassaylateral flow assayReferencesA comparison of standard cultural methods for the detection of foodborne Salmonella / J. Y. D’Aoust [et al.] // Int. J. Food Microbiol. – 1992. – Vol. 16, № 1. – P. 41–50. https://doi.org/10.1016/0168-1605(92)90124-lD’Aoust J. Y., Sewell A. M., Warburton D. W. A comparison of standard cultural methods for the detection of foodborne Salmonella. International Journal of Food Microbiology, 1992, no. 16, vol. 1, pp. 41–50. https://doi.org/10.1016/0168-1605(92)90124-lСоколов, Д. М. Ускоренные методы выявления бактерий рода Salmonella в пищевых продуктах и сырье. / Д. М. Соколов, М. С. Соколова // Вопр. питания. – 2013. – № 1. – С. 33–40.Sokolov D. M., Sokolov M. S. Rapid methods for the genus Salmonella bacteria detection. Voprosy pitaniya = Problems of Nutrition, 2013, no. 1, pp. 33–40 (in Russian).Choi, D. Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of Salmonellas / D. Choi, R. S. Tsang, M. H. Ng // J. Appl. Bacteriol. – 1992. – Vol. 72, № 2. – P. 134–138. https://doi.org/10.1111/j.1365-2672.1992.tb01814.xChoi D., Tsang R. S., Ng M. H. Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of Salmonellas. Journal of Applied Bacteriology, 1992, vol. 72, no. 2, pp. 134–138. https://doi.org/10.1111/j.1365-2672.1992.tb01814.xMonoclonal antibody-based cross-reactive sandwich ELISA for the detection of Salmonella spp. in milk samples / X. Wu [et al.] // Anal. Methods. – 2015. – Vol. 7, № 21. – P. 9047–9053. https://doi.org/10.1039/C5AY01923KWu X., Wang W., Liu L., Kuang H., Xu C. Monoclonal antibody-based cross-reactive sandwich ELISA for the detection of Salmonella spp. in milk samples. Analytical Methods, 2015, vol. 7, no. 21, pp. 9047–9053. https://doi.org/10.1039/C5AY01923KGold nanoparticle-based strip sensor for multiple detection of twelve Salmonella strains with a genus-specific lipopolysaccharide antibody / W. Wang [et al.] // Sci. China Mater. – 2016. – Vol. 59, № 8. – P. 665–674. https://doi.org/10.1007/s40843-016-5077-0Wang W., Liu L., Song S., Xu L., Kuang H., Zhu J., Xu C. Gold nanoparticle-based strip sensor for multiple detection of twelve Salmonella strains with a genus-specific lipopolysaccharide antibody. Science China Materials, 2016, vol. 59, no. 8, pp. 665–674. https://doi.org/10.1007/s40843-016-5077-0A review of methods for the detection of pathogenic microorganisms / P. Rajapaksha [et al.] // Analyst. – 2019. – Vol. 144, № 2. – P. 396–411. https://doi.org/10.1039/c8an01488dRajapaksha P., Elbourne A., Gangadoo S., Brown, R., Cozzolino D., Chapman J. A Review of methods for the detection of pathogenic microorganisms. Analyst, 2019, vol. 144, no. 2, pp. 396–411. https://doi.org/10.1039/c8an01488dAn enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D Salmonellae: a rapid screening prototype/ J. M. Luk [et al.] // J. Clin. Microbiol. – 1997. – Vol. 35, № 3. – P. 714–718. https://doi.org/10.1128/jcm.35.3.714-718.1997Luk J. M., Kongmuang U., Tsang R. S., Lindberg A. A. An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D Salmonellae: a rapid screening prototype. Journal of Clinical Microbiology, 1997, vol. 35, no. 3, pp. 714–718. https:// doi.org/10.1128/jcm.35.3.714-718.1997.Qualitative PCR-ELISA protocol for the detection and typing of viral genomes / M. Musiani [et al.] // Nat. Protoc. – 2007. – Vol. 2, № 10. – P. 2502–2510. https://doi.org/10.1038/nprot.2007.311Musiani M., Venturoli S., Gallinella G., Zerbini M. Qualitative PCR-ELISA protocol for the detection and typing of viral genomes. Nature Protocols, 2007, vol. 2, no. 10, pp. 2502–2510. https://doi.org/10.1038/nprot.2007.311Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria / H.-B. Liu [et al.] // J. Dairy Sci. – 2017. – Vol. 100, № 9. – P. 7016–7025. https://doi.org/10.3168/jds.2017-12566Liu H.-B., Zang Y.-X., Du X.-J., Li P., Wang S. Development of an isothermal amplification-based assay for the rapid visual detection of Salmonella bacteria. Journal of Dairy Science, 2017, vol. 100, no. 9, pp. 7016–7025. https://doi.org/10.3168/jds.2017-12566Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks / J. Hu [et al.] // J. Microbiol. Methods. – 2019. – Vol. 158. – P. 25–32. https://doi.org/10.1016/j.mimet.2019.01.018Hu J., Huang R., Sun Y., Wei X., Wang Y., Jiang C., Geng Y., Sun X., Jing J., Gao H., Wang Z., Dong C. Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification with lateral flow dipsticks. Journal of Microbiological Methods, 2019, vol. 158, pp. 25–32. https://doi.org/10.1016/j.mimet.2019.01.018Europium as a label in time-resolved immunofluorometric assays / I. Hemmilä [et al.] // Anal. Biochem. – 1984. – Vol. 137, № 2. – P. 335–343. https://doi.org/10.1016/0003-2697(84)90095-2Hemmilä I., Dakubu S., Mukkala V. M., Siitari H., Lövgren T. Europium as a label in time-resolved immunofluorometric assays. Analytical Biochemistry, 1984, vol. 137, no. 2, pp. 335–343. https://doi.org/10.1016/0003-2697(84)90095-2Гарбуз, О. С. Лантанидный иммунофлуориметрический анализ: научные основы и технические принципы / О. С. Гарбуз, О. В. Свиридов // ARSmedica. – 2011. – № 13. – С. 51–61.Garbuz O. S., Sviridov O. V. Lanthanide immunofluorimetric assay: scientific background and technical principles. ARSmedica, 2011, no. 13, pp. 51–61 (in Russian).Функционализированные металлохелаты на основе диэтилентриаминтетрауксусной кислоты для химической модификации белков и малых биомолекул / О. С. Куприенко [и др.] // Биоорг. химия. – 2015. – Т. 41, № 6. – С. 675–685. https://doi.org/10.7868/S013234231506007XKuprienko O. S., Dubovskaya L. V., Shabunya P. S., Fatyhova S. A., Sviridov O. V. Functionalized metal chelates based on diethylenetriaminetetetraacetic acids for chemical modification of proteins and small biomolecules. Bioorganicheskaja himiya. = Bioorganic Chemistry, 2015, vol. 41, no. 6, pp. 675–685 (in Russian). https://doi.org/10.7868/S013234231506007XFrens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions / G. Frens // Nat. Phys. Sci. – 1973. – Vol. 241, № 105. – P. 20–22. https://doi.org/10.1038/physci241020a0Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Physical Science, 1973, vol. 241, no. 105, pp. 20–22. https://doi.org/10.1038/physci241020a0Lateral flow immunoassay for rapid qualitative and quantitative control of the veterinary drug bacitracin in milk / N. A. Byzova [et al.] // Microchem. J. – 2020. – Vol. 156. – Article 104884. https://doi.org/10.1016/j.microc.2020.104884Byzova N. A., Serchenya T. S., Vashkevich I. I., Zherdev A. V., Sviridov O. V., Dzantiev B. B. Lateral flow immunoassay for rapid qualitative and quantitative control of the veterinary drug bacitracin in milk. Microchemical Journal, 2020, vol. 156, article 104884. https:// doi.org/10.1016/j.microc.2020.104884Hermanson, G. T. Bioconjugate Techniques / G. T. Hermanson. – Elsevier, 1996. – 814 p. https://doi.org/10.1016/0890-8508(92)90002-fHermanson G. T. Bioconjugate Techniques. Elsevier, 1996. 814 p. https://doi.org/10.1016/0890-8508(92)90002-fGalán, J. E. Cloning and molecular characterization of genes whose products allow Salmonella Typhimurium to penetrate tissue culture cells / J. E. Galán, R. Curtiss // Proc. Nat. Acad. Sci. USA. – 1989. – Vol. 86, № 16. – P. 6383–6387. https://doi.org/10.1073/pnas.86.16.6383Galán J. E., Curtiss R. Cloning and molecular characterization of genes whose products allow Salmonella Typhimurium to penetrate tissue culture cells. Proceedings of the National Academy of Sciences of the United States of America, 1989, vol. 86, no. 16, pp. 6383–6387. https://doi.org/10.1073/pnas.86.16.6383Amplification of an invA gene sequence of Salmonella Typhimurium by polymerase chain reaction as a specific method of detection of Salmonella / K. Rahn [et al.] // Mol. Cell. Probes. – 1992. – Vol. 6, № 4. – P. 271–279. https://doi.org/10.1016/0890-8508(92)90002-fRahn K., De Grandis S. A., Clarke R. C., McEwen S. A., Galán J. E., Ginocchio C., Curtiss R., Gyles C. L. Amplification of an invA gene sequence of Salmonella Typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Molecular and Cellular Probes, 1992, vol. 6, no. 4, pp. 271–279. https://doi.org/10.1016/0890-8508(92)90002-fNucleic acid lateral flow assay with recombinase polymerase amplification: solutions for high-sensitive detection of RNA virus // A. V. Ivanov [et al.] // Talanta. – 2020. – Vol. 210. – Article 120616. https://doi.org/10.1016/j.talanta.2019.120616Ivanov A. V., Safenkova I. V., Zherdev A. V., Dzantiev B. B. Nucleic acid lateral flow assay with recombinase polymerase amplification: solutions for high-sensitive detection of RNA virus. Talanta, 2020, vol. 210, article 120616. https://doi.org/10.1016/j.talanta.2019.120616Moon, Y.-J. A review of isothermal amplification methods and food-origin inhibitors against detecting food-borne pathogens / Y.-J. Moon, S.-Y. Lee, S.-W. Oh // Foods. – 2022. – Vol. 11, № 3. – P. 322–337. https://doi.org/10.3390/foods11030322Moon Y.-J., Lee S.-Y., Oh S.-W. A review of isothermal amplification methods and food-origin inhibitors against detecting foodborne pathogens. Foods, 2022, vol. 11, no. 3, pp. 322–337. https://doi.org/10.3390/foods11030322An оverview for the nanoparticles-based quantitative lateral flow assay // Z. Wang [et al.] // Small methods. – 2022. – Vol. 6, № 1. – Article 2101143. https://doi.org/10.1002/smtd.202101143Wang Z., Zhao J., Xu X., Guo L., Xu L., Sun M., Hu S., Kuang H., Xu C., Li A. An Overview for the nanoparticles-based quantitative lateral flow assay. Small methods, 2022, vol. 6, no. 1, article 2101143. https://doi.org/10.1002/smtd.202101143

The authors declare that there are no conflicts of interest present.