Application of molecular methods for microbial identifi cation in dairy products

Abstract

© 2015 by Taylor & Francis Group, LLC. Although traditional approaches such as cultivation, physiological and chemo taxonomic methods are the cornerstone of the isolation and characterization of individual organisms and complex communities; molecular methods have made, and continue to make, incredible contributions to the study of microbial diversity. A major advantage of molecular methods is the ability to process large numbers of samples simultaneously and have been termed high-throughput methods. Principle investigators and students alike, therefore, favor such methods due to the huge amount of data that can be generated in a relatively short period of time in a cost-effective manner. Indeed many would argue that molecular methods have surpassed the more traditional methods, but this viewpoint is unwise. No one method can answer all questions and even the most powerful approaches such as genome analysis must be complemented by physiological investigations to provide a comprehensive polyphasic approach (Rainey 2011). For example, a gene may well be present in the genome but is it expressed at all, and if so, when? These questions are particularly important when considering the microbial community as a whole. Therefore, it is essential that high-throughput molecular methods be used in tandem with more traditional methods for a comprehensive investigation of microorganisms present and their potential roles in food spoilage as food pathogens, food additives, etc. With respect to food-borne organisms and the associated pathogens, in addition to cultivation and enzyme-linked immunosorbent assay (ELSA) three main approaches using molecular tools may be employed. The first are methods based on the polymerase chain reaction (PCR) (Hayden 2004) with Escherichia coli (Tsai et al. 1993, Naravaneni and Jamil 2005) Salmonella (Rahn et al. 1992) Shigella (Frankel et al. 1990) Yersinia (Ibrahim et al. 1992) Vibrio cholera (Shangkuan et al. 1995), Vibrio parahaemolyticus (Tada et al. 1992) Vibrio vulnificus (Brauns et al. 1991), Listeria monocytogenes (Simon et al. 1996), and Staphylococcus aureus (Wilson et al. 1991). A further refinement was the introduction of Real-Time PCR that is now the most commonly used technology for quantification of specific DNA fragments (Wittwer and Kusukawa 2004). The amount of product synthesized during the PCR is measured in real time by detection of the fluorescent signal produced as a result of specific amplification. The PCR methods are rapid and sensitive, but care should be taken with appropriate controls as false-positive and false-negative results can lead to misleading conclusions.