Abstract

Background: The dairy processing industry is currently hindered by slow, antiquated testing for high-risk pathogens, including Listeria monocytogenes and Cronobacter sakazakii. Powder infant formula's low water activity and substantial fat content enhance the survival and thermotolerance of such foodborne pathogens. As DNA-based detection methods can produce false positives even after cell death, a more accurate approach is needed. The objective of this study is to evaluate the suitability of an RNA-based platform for the detection of pathogens in a dairy powder matrix. Materials and Methods: A unique phenol-chloroform extraction method was used to obtain total RNA from heat-killed and unheated cells, and an adapted version of the same method allowed for extraction from reconstituted PIF. mRNA was quantified using RT-qPCR and rRNA was visualized on agarose gels. Fold change values were calculated from the raw Ct values of qPCR. Results: Our findings showed unexpected stability of mRNA in heat-killed (72Â°C for 10 minutes) C. sakazakii and L. monocytogenes that were incubated at room temperature in a preferred growth medium (BHI) for 48 hours, with statistically insignificant fold changes. During the same incubation period, mRNA levels in live (control) cells diminished almost entirely. Contrastingly, the same transcripts displayed different behavior in reconstituted PIF. In heat-killed cells, the mRNA transcripts were detected in low quantities and degraded rapidly in the dairy matrix, while those in unheated formula experienced less degradation over time. Conclusions: The rapid degradation of mRNA in heat-killed C. sakazakii and L. monocytogenes in PIF suggests its promising suitability as a pathogen detection analyte. This study demonstrates that mRNA degradation under these circumstances is both transcript and matrix specific. The unexpected persistence of mRNA transcripts after thermal inactivation in a BHI medium challenges the previously assumed instability of mRNA.

Degree

College and Department

Life Sciences; Nutrition, Dietetics, and Food Science

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