title: Critical issues in detecting viable Listeria monocytogenes cells by real-time reverse transcriptase PCR.
abstract: Rapid and specific detection of viable Listeria monocytogenes cells, particularly in processed foods, is a major challenge in the food industry. To assess the suitability of using RNA-based detection methods to detect viable cells, several sets of PCR primers and florescent probes  were designed targeting the 16S rRNA, internalin A, and ribosomal protein L4 genes. One-step real-time reverse transcriptase (RT) PCR assays were conducted using RNAs extracted from control and heat-treated L. monocytogenes samples. The cycle threshold values were significantly higher  in heat-treated cells than in controls. However, real-time RT-PCR amplification signals were still detected even in samples stored at room temperature for 24 h after lethal treatments, and the intensity of the signals was correlated with the cell population. The 16S rRNA molecules were the  most stable of the three targets evaluated, and the impact on detection efficacy of the relative positions of the PCR primers within the target genes was limited under the experimental conditions. These results suggest that real-time RT-PCR assays have advantages over conventional PCR assays  for assessing viable L. monocytogenes cells, but the results are affected by the stability of the RNA molecules targeted. These findings could have a major impact on interpretation of RNA-based detection data and gene expression studies.
authors.affiliation: Department of Food Science, The Ohio State University, 2015 Fyffe Court, Columbus, Ohio 43210, USA.
authors.forename: Linlin
authors.initials: L
authors.lastname: Xiao
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.forename: Hua H
authors.initials: HH
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D003257:Consumer Product Safety; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S