Challenges to developing nucleic acid sequence based amplification technology for the detection of microbial pathogens in food

Microbiological quality control programs are increasingly applied through out food chain production in order to minimize the risk of foodborne diseases for the consumer. At present, traditional and standardized analysis of foods have low limits of detection and can be used in complex food sample matrices, but they are labour-intensive and typically require days from initiation to readout. Nucleic acid sequence-based amplification (NASBA) has been used to achieve RNA-based technology as an alternative method. Indeed, this technology not only enhances the speed of detection, but the specificity of the probe also could eliminate the requirement for additional confirmation. NASBA has shown great promise as highly sensitive and specific tool for the amplification and detection of pathogen-specific RNA sequences of Compylobacter jejuni, Listeria monocytogenes, Salmonella enterica, Bacillus spp. and Escherichia coli in foods. The different protocols applied in these studies rely on different steps: (i) concentrating and separating bacteria from the sample matrix, (ii) RNA preparation, (iii) NASBA amplification; and (iv) post NASBA detection. In the future, NASBA could be used to identify and subsequently quantify microorganisms (even those which cannot be readily cultured) and would be very efficient as routine diagnostic procedures.