Combination of Immunomagnetic Separation and Real-Time Recombinase Polymerase Amplification (IMS-qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples

摘要

Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real-time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real-time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 x 10(2) cfu/25 g. L. monocytogenes was detected after a 24 hr pre-enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD(50)) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as "almost complete concordance" between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real-time PCR. Practical Application The methodology described in this study significantly reduces the detection time of L. monocytogenes, when compared with culture-based methods, and it requires fewer steps than other molecular methods, making it a reliable and more convenient method for routine testing. Finally, the evaluation of the methodology in spiked food samples, confirms its reliability.