Development and evaluation of a loop-mediated isothermal amplification assay (lamp) for the diagnosis of campylobacteriosis
- Authors: Shuryaeva A.K.1, Malova T.V.1, Tolokonceva A.A.1, Karseka S.A.1, Davydova E.E.1, Shipulin G.A.1
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Affiliations:
- Centre for Strategic Planning and Management of Biomedical Health Risks
- Issue: Vol 12, No 3 (2021)
- Pages: 30-35
- Section: Original Study Articles
- URL: https://journals.rcsi.science/clinpractice/article/view/78139
- DOI: https://doi.org/10.17816/clinpract78139
- ID: 78139
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Abstract
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 65°C 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.
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##article.viewOnOriginalSite##About the authors
Anna K. Shuryaeva
Centre for Strategic Planning and Management of Biomedical Health Risks
Author for correspondence.
Email: ashuryaeva@cspmz.ru
ORCID iD: 0000-0003-2227-1840
Junior Research Associate
Russian Federation, 15A, building 54, 3rd Cherepkovskaya street, 121552, MoscowTatyana V. Malova
Centre for Strategic Planning and Management of Biomedical Health Risks
Email: TMalova@cspmz.ru
ORCID iD: 0000-0001-8546-1444
Junior Research Associate
Russian Federation, MoscowAnna A. Tolokonceva
Centre for Strategic Planning and Management of Biomedical Health Risks
Email: ATolokonceva@cspmz.ru
ORCID iD: 0000-0002-8757-081X
Biologist
Russian Federation, MoscowSofia A. Karseka
Centre for Strategic Planning and Management of Biomedical Health Risks
Email: SKarseka@cspmz.ru
ORCID iD: 0000-0002-9988-6918
Laboratory Assistant
Russian Federation, MoscowEkaterina E. Davydova
Centre for Strategic Planning and Management of Biomedical Health Risks
Email: EDavydova@cspmz.ru
ORCID iD: 0000-0003-2926-0490
Cand. Sci. (Chem.)
Russian Federation, MoscowGerman A. Shipulin
Centre for Strategic Planning and Management of Biomedical Health Risks
Email: shipgerman@gmail.com
ORCID iD: 0000-0002-3668-6601
MD, Cand. Sci. (Med.)
Russian Federation, MoscowReferences
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