ABSTRACT
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
1 Department of Microbiology and Plant Biology, University of Oklahoma, OK 73019, USA. Email: paul.lawson@ou.edu 2 Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University
of Technology, P.O. Box 50329, 3603 Limassol, CYPRUS. Email: dimitris.tsaltas@cut.ac.cy * Corresponding author
so, when? These questions are particularly important when considering the microbial community as a whole. Therefore, it is essential that highthroughput 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 fi rst 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 vulnifi cus (Brauns et al. 1991), Listeria monocytogenes (Simon et al. 1996), and Staphylococcus aureus (Wilson et al. 1991). A further refi nement was the introduction of Real-Time PCR that is now the most commonly used technology for quantifi cation of specifi c DNA fragments (Wittwer and Kusukawa 2004). The amount of product synthesized during the PCR is measured in real time by detection of the fl uorescent signal produced as a result of specifi c amplifi cation. 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.
