APPLICATION OF THE END POINT POLYMERASE CHAIN REACTION METHOD TO THE DETECTION OF CERTAIN MILK-BORNE BACTERIA

The polymerase chain reaction (PCR) is a modern method that could differentiate microorganisms from each other by detecting certain DNA sequences uniquely associated with each species. This study aimed to develop a relevant end point PCR assay to detect Salmonella ser. Typhimurium in milk. To pursue this objective, the conditions of both DNA extraction and PCR reactions were developed in terms of the boiling time for DNA extraction and primer concentration for the PCR assay. Results showed that all of the three examined boiling times of 10, 15 and 20 min were equally effective for carrying out efficient DNA extraction. PCR reaction mixture involving a primer concentration of 1300 nM was the most efficient concentration, compared to 650 nM, and 325 nM for detecting Salmonella ser. Typhimurium. The sensitivity of the developed PCR method to detect different viable numbers of Salmonella Typhimurium in tryptone soya broth (TSB) and reconstituted skim milk (RSM) was examined. The PCR assay was able to detect 10^9, 10^8, 10^7, and 10^6 cfu mL^-1 of Salmonella in TSB. Whereas, it could detect 10^9, and 10^8 cfu mL^-1 of Salmonella in RSM. These results suggested that the developed PCR method had higher sensitivity to detect Salmonella in TSB, compared to RSM. This was attributed to some milk components that could be inhibitory to PCR reactions.