Molecular Methods in Food Safety Microbiology: Interpretation and Implications of Nucleic Acid Detection

Abstract

Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the associated public health risk. Discrepancies between results obtained by molecular and conventional culture methods stem from the difference in target, namely nucleic acids instead of actively growing microorganisms. Nucleic acids constitute 5% to 15% of the dry weight of all living cells and are relatively stable, even after cell death, so they may be present in a food matrix after the foodborne microorganisms have been inactivated. Therefore, interpretation of the public health significance of positive results generated by nucleic acid detection methods warrants some additional consideration. This review discusses the stability of nucleic acids in general and highlights the persistence of microbial nucleic acids after diverse food-processing techniques based on data from the scientific literature. Considerable amounts of DNA and RNA (intact or fragmented) persist after inactivation of bacteria and viruses by most of the commonly applied treatments in the food industry. An overview of the existing adaptations for molecular assays to cope with these problems is provided, including large fragment amplification, flotation, (enzymatic) pretreatment, and various binding assays. Finally, the negligible risks of ingesting free microbial nucleic acids are discussed and this review ends with the future perspectives of molecular methods such as next-generation sequencing in diagnostic and source attribution food microbiology.

Thermal Inactivation of Mycobacterium avium subsp. paratuberculosis in Artificially Contaminated Milk by Direct Steam Injection

“…paratuberculosis DNA was only mildly degraded at such high thermal treatments, resulting in higher C T values post-DSI. Our findings confirm previous reports in which it was shown that DNA can withstand thermal degradation to yield positive PCR results (43,44). More importantly, the qPCR signal obtained post-DSI may explain the higher M. avium subsp.…”

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage

“…However, in some cases, EMA and PMA could also diffuse into living bacterial cells with intact membranes (14). On the other hand, RNA is considered a more useful target for viable bacteria even though rRNA molecules remain available for detection after bacterial death for some generally not predictable time (14). Moreover, RNA should be considered a more informative target than DNA, as rRNA can be proportionally more abundant in microbial cells than DNA copies, and this could lead to a more detailed picture of the matrix analyzed (15).…”

“…To avoid this bias, several authors suggested the use of treatments with propidium monoazide (PMA) or ethidium monoazide (EMA) prior to DNA extraction to detect or quantify viable microorganisms. However, in some cases, EMA and PMA could also diffuse into living bacterial cells with intact membranes (14). On the other hand, RNA is considered a more useful target for viable bacteria even though rRNA molecules remain available for detection after bacterial death for some generally not predictable time (14).…”

High-Throughput Sequencing Technologies and new Approaches in Food Microbiology

“…Most of these pathogens are not easily individuated by using traditional approaches, and metagenomic methods have been adopted, mainly using 16s rDNA or 26s rDNA gene sequencing [16,17]. These approaches have a growing importance also in the authentication product activity [18].…”