Next-day detection of viable Listeria monocytogenes by multiplex reverse transcriptase real-time PCR

Abstract

Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.