May 2017 Vol. 5 No.5

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Original Research Article

Advantages of Eva Green real-time mPCR compared to culture methods for differentiating C. jejuni / coli directly from feces

Maria Pavlova¹, Veleri Veliv^2*, Elina Dobreva¹, Galina Asseva¹, Atanas Mangarov², Ivelina Tomova² and Todor Kantardjiev¹

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

*Corresponding Author’s E-mail: velev_md@avb.bg

Accepted May 01, 2017

Abstract

The objective of this study is 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. 38 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) (CerTestBiotec). 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. Out of 38 clinical fecal samples which were ICT positive, 18 strains were isolated by culture – 17 of C. jejuni and 1 of C. coli. The Eva Green real-time mPCR reaction also reported 18 positive samples for Campylobacter – 17 out of which were of C. jejuni and only one of C.coli. All other samples were negative for Campylobacter spp. The analytical sensitivity and specificity of the mPCR method were 100%. 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.

Keywords: Campylobacter, diarrhea, mPCR