Comparison of DNA amplification methods for simultaneous detection of Salmonella spp. and Cronobacter sakazakii in milk

Abstract

The control and detection of foodborne pathogens is a major problem in public health. Therefore, the development of rapid and economical diagnostic method along the food chain has become a priority. Among the available methodologies, those based on the detection of nucleic acids provide certain advantages such as high sensitivity speed, selectivity, and the ability to simultaneously detect multiple microorganisms. However, the amount of pathogen DNA present in a food is minimal, so these methods must incorporate an amplification stage that achieves the proper amount for their detection. The polymerase chain reaction (PCR) is the common technique, although this method has the requirement of thermocycling. In this master project two enzymatic reactions that enable the amplification of DNA under isothermal conditions have been studied. The work focuses on the comparison of conventional PCR with two isothermal amplification methods ‐recombinase polymerase (RPA) and multiple displacement amplification (MDA)‐. These methods, applied to the detection of Salmonella spp. and Cronobacter sakazakii, have shown excellent amplification yields of the target sequences. In addition, the inhibitory effect of some matrix components on the enzymes has been studied. The simultaneous detection of two pathogens has been made in milk samples, analyzing the amplified products by hybridization assays with oligonucleotide probes immobilized on the surface of a DVD in microarray format. The results have been satisfactory in terms of reproducibility and sensitivity. It has been shown that the use of other alternative enzymes and low cost heating equipments allow DNA amplification at constant temperature. Moreover, the combination with compact disc technology increases the advantages, demonstrating the validity of the proposed biosensor in routine applications in the area of food safety.