ADVANTAGES OF EVA GREEN REAL-TIME MPCR WITH CULTURE AND IMMUNOCHROMATO- GRAPHIC METHODS FOR DIFFERENTIATING C. JEJUNI / COLI DIRECTLY FROM FECES

MARIA PAVLOVA¹, VALERI VELEV², ELINA DOBREVA¹, GALINA ASSEVA¹, IVAN N. IVANOV¹, IVELINA TOMOVA², TODOR KANTARDJIEV¹

¹National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria - ²Department of Infectious Diseases and Parasitology, Medical University of Sofia, Bulgaria

Objective: To develop and optimize a rapid molecular method for diagnosing campylobacteriosis directly from a clinical fecal sample and at the same time determining the most common causing agents - C. jejuni/coli.

Materials and methods: 40 clinical fecal samples of hospitalized patients in the Pediatric Clinic of Infectious Diseases - Sofia were tested, the patients aged between 0 and 7 years and with diarrheal syndrome. The clinical samples were tested using a rapid immunochromatographic test (ICT) (CerTest Biotec). All positive samples were tested for confirmation by culturing in a microaerophilic atmosphere at 42 °C and subsequently the isolates were biochemically differentiated. The Eva Green real-time mPCR reaction of a direct fecal sample was conducted using the “IQ5TM Real-Time PCR System” apparatus.

Results: Out of 40 clinical fecal samples which were ICT positive, 20 strains were isolated by culture – 18 of C. jejuni and 2 of C. coli. The Eva Green real-time mPCR reaction also reported 20 positive samples for Campylobacter – 18 out of which were of C. jejuni and 2 of C.coli. All other samples were negative for Campylobacter spp. The analytical sensitivity and specificity of the mPCR method were 100%.

Discussion: We developed and optimized the Eva Green real-time mPCR for detection and species differentiation of C. jejuni/coli directly from a clinical fecal sample. This analysis ensures the faster and more reliable detection of bacterial cells when compared to the conventional culture methods using biochemical differentiation.