Rapid detection of viable Listeria monocytogenes in chilled pork by real-time reverse-transcriptase PCR.

摘要

The objective of this study was to develop an RNA-dependent real-time reverse-transcriptase PCR (real-time RT-PCR) method for the detection of Listeria monocytogenes in chilled pork without the need for pre-enrichment steps, and the soundness of the method was simultaneously validated and evaluated by DNA-based real-time PCR and traditional culture methods. For specificity testing, a lack of amplification signals and no Tm peak at ~78.37 degrees C were obtained from any of 41 other bacterial strains associated with meat species under the conditions used. The R2 and efficiency of standard curves constructed by ten-fold serial dilutions of pure L. monocytogenes were respectively 0.995 and 90.1%; lower than that of the DNA-based assay. The detection limit was up to 100 cfu/mL in both pure culture and in artificially contaminated chilled pork samples. Quantitative detection showed that the RNA-based assay obtained relatively accurate results when samples had undergone treatments (such as high pressure), but without treatment, the results showed a slight deviation compared with plate counts. The RNA-dependent real-time RT-PCR method developed in this study was found to be rapid and sensitive and should be useful for reliable detection of viable L. monocytogenes in chilled pork, especially for pre-treated samples. However, this method cannot be recommended to accurately quantify L. monocytogenes, but only to show the presence of live cells and approximately predict its level of contamination.