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

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.

Keywords

gastrointestinal infections / molecular diagnostics / rapid diagnostics / Loop-Mediated Isothermal Amplification / LAMP / Campylobacter spp.

References

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Funding

Государственное задание «Разработка экспресс-тестов для диагностики острых кишечных инфекционных заболеваний методом изотермической амплификации»State task «Development of rapid tests for the diagnosis of acute intestinal infectious diseases by the method of isothermal amplification»

RIGHTS & PERMISSIONS

Shuryaeva A.K., Malova T.V., Tolokonceva A.A., Karseka S.A., Davydova E.E., Shipulin G.A.