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 causes of bacterial gastroenteritis. There are many methods to detect the presence of Campylobacter, including PCR, but it takes about 5-6 hours. Using loop-mediated amplification assay allowed reducing the time of detection and simplifying the procedure at all.
 Aims: To develop a loop-mediated isothermal amplification assay (LAMP) with fluorescent probe for the diagnosis of campylobacteriosis.
 Methods: Stool suspensions were prepared and bacterial fractions were separated as in methodological recommendation of Central Research Institute of Epidemiology described. DNA was extracted using AmpliTest RIBO-prep (FSBI SPC FMBA, Russian Federation) according to the manufacturers instruction and detected with AmpliSens OKI-screen-FL" (FBIS CRIE, Russian Federation). Primers and probes were selected in 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 is 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 the 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 is 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.