Magnetic nanoparticle based PCR assay for detection of Campylobacter species in food samples and dielectrophoretic impedance measurement for DNA detection

Abstract

Campylobacter species are major foodborne pathogens, causing bacterial gastroenteritis worldwide. Poultry are considered to be important reservoirs of Campylobacter transmission and campylobacteriosis commonly relates to the consumption of undercooked chicken products. This study successfully developed a magneto-PCR-enzyme linked gene assay for the detection of thermophilic Campylobacter from chicken skin. The forward primers immobilized on magnetic nanoparticles captured Campylobacter DNA to be amplified by PCR reaction and the PCR products were detected by streptavidin-horseradish peroxidase and its substrate. The method demonstrated 100% specificity and high sensitivity with one picogram detection of Campylobacter DNA. All positive results derived from the detection in naturally contaminated samples were in accordance with the results analyzed by a conventional cultural method and PCR assay. In this study, the dielectrophoretic impedance measurement (DEPIM) method has been used for the detection of PCR and LAMP products by measuring an increment of the conductance. Dielectrophoresis is a motion of the polarized particles in spatially non-uniform electric fields. The DEP- trapping process of DNA, generated from PCR or LAMP products, was not visualized by a fluorescence microscopy. However, in an AC electric field of 150 kHz, the conductance increased when applying the undiluted PCR product of 187 base pairs. After optimizing conditions for detecting LAMP product, only undiluted and ten-fold diluted LAMP products significantly demonstrated the conductance increment. This preliminary study showed the possibility of applying The DEPIM method for detection of PCR and LAMP products.