Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis

Background: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplific...

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Autores principales:	Anna K. Shuryaeva, Tatyana V. Malova, Anna A. Tolokonceva, Sofia A. Karseka, Ekaterina E. Davydova, German A. Shipulin
Formato:	article
Lenguaje:	RU
Publicado:	Eco-vector 2021
Materias:	gastrointestinal infections molecular diagnostics rapid diagnostics loop-mediated isothermal amplification lamp campylobacter spp. Medicine R
Acceso en línea:	https://doaj.org/article/51e0539f37bd4259bbbcd0059720f3c3
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spelling	oai:doaj.org-article:51e0539f37bd4259bbbcd0059720f3c32021-11-30T18:15:22ZDevelopment and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis2220-30952618-862710.17816/clinpract78139https://doaj.org/article/51e0539f37bd4259bbbcd0059720f3c32021-10-01T00:00:00Zhttps://journals.eco-vector.com/clinpractice/article/viewFile/78139/pdfhttps://doaj.org/toc/2220-3095https://doaj.org/toc/2618-8627Background: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplification assay will allow simplifying the procedure and reducing the time of detection for a bedside application. Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with a fluorescent probe for the diagnosis of campylobacteriosis. Methods: Stool suspensions were prepared and bacterial fractions were separated as described in the methodological recommendations of the Central Research Institute of Epidemiology. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturer's instruction and detected with AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation). Primers and probes were selected in a 16S rDNA gene region. Analytical specificity was confirmed on bacterial cultures, analytical sensitivity was assessed using a recombinant plasmid containing the target Campylobacter DNA sequence fragment. LAMP amplification was performed at 65C for 30 min. Results: An assay for the detection of Campylobacter spp. based on loop-mediated isothermal amplification has been developed, the reaction time does not exceed 30 minutes. The analytical sensitivity of the developed technique is comparable to the real-time PCR and is equal to 103 copies/ml, the analytical specificity is 100%. The evaluation of 127 clinical samples, previously characterized by a commercial kit, AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation), showed high diagnostic specificity and sensitivity of the developed LAMP-method. No false positive results were found, 108 samples were negative by LAMP and PCR. Campylobacter spp. DNA was detected by the LAMP method in 18 out of 19 PCR-positive samples. One discordant LAMP negative sample can be attributed to the low bacterial load of Campylobacter spp. for a given sample. Conclusions: A method for the rapid detection of Campylobacter spp. loop-mediated isothermal amplification has been developed, and its high analytical and diagnostic characteristics have been shown experimentally.Anna K. ShuryaevaTatyana V. MalovaAnna A. TolokoncevaSofia A. KarsekaEkaterina E. DavydovaGerman A. ShipulinEco-vectorarticlegastrointestinal infectionsmolecular diagnosticsrapid diagnosticsloop-mediated isothermal amplificationlampcampylobacter spp.MedicineRRUКлиническая практика , Vol 12, Iss 3, Pp 30-35 (2021)
institution	DOAJ
collection	DOAJ
language	RU
topic	gastrointestinal infections molecular diagnostics rapid diagnostics loop-mediated isothermal amplification lamp campylobacter spp. Medicine R
spellingShingle	gastrointestinal infections molecular diagnostics rapid diagnostics loop-mediated isothermal amplification lamp campylobacter spp. Medicine R Anna K. Shuryaeva Tatyana V. Malova Anna A. Tolokonceva Sofia A. Karseka Ekaterina E. Davydova German A. Shipulin Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
description	Background: Different species of Campylobacter are the most common cause of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes no less than 5 -6 hours. Development of fast molecular diagnostic tests based on a loop-mediated amplification assay will allow simplifying the procedure and reducing the time of detection for a bedside application. Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with a fluorescent probe for the diagnosis of campylobacteriosis. Methods: Stool suspensions were prepared and bacterial fractions were separated as described in the methodological recommendations of the Central Research Institute of Epidemiology. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturer's instruction and detected with AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation). Primers and probes were selected in a 16S rDNA gene region. Analytical specificity was confirmed on bacterial cultures, analytical sensitivity was assessed using a recombinant plasmid containing the target Campylobacter DNA sequence fragment. LAMP amplification was performed at 65C for 30 min. Results: An assay for the detection of Campylobacter spp. based on loop-mediated isothermal amplification has been developed, the reaction time does not exceed 30 minutes. The analytical sensitivity of the developed technique is comparable to the real-time PCR and is equal to 103 copies/ml, the analytical specificity is 100%. The evaluation of 127 clinical samples, previously characterized by a commercial kit, AmpliSens OKI-screen-FL (FBIS CRIE, Russian Federation), showed high diagnostic specificity and sensitivity of the developed LAMP-method. No false positive results were found, 108 samples were negative by LAMP and PCR. Campylobacter spp. DNA was detected by the LAMP method in 18 out of 19 PCR-positive samples. One discordant LAMP negative sample can be attributed to the low bacterial load of Campylobacter spp. for a given sample. Conclusions: A method for the rapid detection of Campylobacter spp. loop-mediated isothermal amplification has been developed, and its high analytical and diagnostic characteristics have been shown experimentally.
format	article
author	Anna K. Shuryaeva Tatyana V. Malova Anna A. Tolokonceva Sofia A. Karseka Ekaterina E. Davydova German A. Shipulin
author_facet	Anna K. Shuryaeva Tatyana V. Malova Anna A. Tolokonceva Sofia A. Karseka Ekaterina E. Davydova German A. Shipulin
author_sort	Anna K. Shuryaeva
title	Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
title_short	Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
title_full	Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
title_fullStr	Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
title_full_unstemmed	Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
title_sort	development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
publisher	Eco-vector
publishDate	2021
url	https://doaj.org/article/51e0539f37bd4259bbbcd0059720f3c3
work_keys_str_mv	AT annakshuryaeva developmentandevaluationofaloopmediatedisothermalamplificationassaylampforthediagnosisofcampylobacteriosis AT tatyanavmalova developmentandevaluationofaloopmediatedisothermalamplificationassaylampforthediagnosisofcampylobacteriosis AT annaatolokonceva developmentandevaluationofaloopmediatedisothermalamplificationassaylampforthediagnosisofcampylobacteriosis AT sofiaakarseka developmentandevaluationofaloopmediatedisothermalamplificationassaylampforthediagnosisofcampylobacteriosis AT ekaterinaedavydova developmentandevaluationofaloopmediatedisothermalamplificationassaylampforthediagnosisofcampylobacteriosis AT germanashipulin developmentandevaluationofaloopmediatedisothermalamplificationassaylampforthediagnosisofcampylobacteriosis
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Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis

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