Rapid Diagnosis of Listeria monocytogenes by PCR Method with hlyA gene

Currently the conventional method for the diagnosis of Listeria monocytogenes is culture method. However, the isolation rate is reduced in culture due to animal's food which is contains antibiotics. Besides, culture method is time consuming and takes more than 36 hrs. So more precise and rapid technique is needed. The aim of this study was application of PCR technique based on hlyA gene fragment for rapid detection of Listeria monocytogenes in clinical samples. hlyA gene was selected as a specific target sequencefor detection of Listeria monocytogenes. Listeria monocytogenes PTCC1163 was used as a standard organism for optimization experiments. A range of bacterial pathogens was used for specificity test including Escherichia coli: ATCC;35218 Salmonella typhi PTCC: 1609. Phenol – chloroform method was used for DNA extraction. PCR technique was performed as per standard protocol. Amplified product was detected by 1% gel agarose electrophoresis, stained by ethidiome bromide. Provided data confirmed amplification of expected product. Specificity test proved no cross reaction with tested organisms. Sensitivity test detected 500fg Listeria monocytogenes DNA as a final detection limit. Optimized experiment confirmed that the applied PCR protocol is quite fast with high sensitivity and specificity performing in less than three hours.Application of this method in clinical laboratories can help the rapid diagnosis of Listeria monocytogenes in samples.