Multiplex PCR for direct identification of Campylobacter Species in Human Stool

Abstract

Background

Campylobacter spp. are a major cause of bacterial gastroenteritis worldwide. Is one of the most frequently isolated bacteria from the feces of infants and children in developing countries, Conventional diagnostic methods for the detection and differentiation of Campylobacter species are tedious and time consuming, we have developed a multiplex PCR protocol using a novel combination of species-specific and virulence genes, The multiplex PCR protocol was capable of detecting the type strains and clinical isolates from all five species with a high degree of specificity.

Methods: Molecular testing for Campylobacter Universal gene was done on 50 Stool specimens using Multiplex PCR technique. Stool specimens were collected from patients suffering from gastroenteritis, and kept at -20 ^OC till used .DNA Extraction was done by using Vivantis GF-1 Nucleic acid extraction kit (Vivantis, MALAYSIA). The amplification reaction was carried out in thermo cycler with program system consisting of ''( Initial denaturation step at 95°C for 6 min followed by 30 cycles of amplification (denaturation at 95°C for 0.5 min, annealing at 59°C for 0.5 min, and extension at 72°C for 0.5 min), ending with a final extension at 72°C for 7 min)'' PCR products were separated in a 1.5% agarose gel, then stained with ethidium bromide and viewed under gel documentation system. A result was considered positive when a band of the appropriate size was visible in the gel. Standard procedures for reducing contamination were strictly followed.

Results: eight samples (16%) out of 50 were positive by Multiplex PCR, while 42 samples (84%) were negative.

Conclusion: The multiplex PCR is very useful for direct detection and diagnosis of campylobacteriosis.