Development of asigDE-Based Real-Time Reverse-Transcriptase PCR for the Detection of ViableSalmonella enterica

Abstract

Salmonella is the most common cause of bacterial food poisoning in humans worldwide. Thus, rapid and reliable methods for the detection of this pathogen are required. Real-time reverse-transcriptase polymerase chain reaction (rt-RT-PCR), which detects the presence of mRNA (shorter half-life than DNA) has shown great potential for detecting viable pathogens. We recently identified a few new potential specific DNA sequences for Salmonella enterica using a comparative genomics method (Chen et al., 2010). In the present study, we examined the expression of the Salmonella-specific sigDE operon (encoding invasion proteins within the pathogenicity island 5) under typical growth conditions to determine whether sigDE could be a useful viability marker for the bacterium. We then assayed sigDE mRNA from cells heat-treated at 60°C, 100°C, and 121°C (autoclaved), and found that mRNA was degraded in autoclaved bacterial samples. These results showed that the sigDE transcript is a suitable mRNA target for rt-RT-PCR with samples pretreated at 121°C. Thus, an rt-RT-PCR using sigDE primers was developed for the detection of viable Salmonella. An RNA internal amplification control was constructed by overlap extension PCR, synthesized using in vitro transcription with a T7 RNA polymerase promoter, and incorporated into the rt-RT-PCR system to eliminate false-negative results. The rt-RT-PCR system has the capability of specifically detecting all the tested S. enterica serovars, and the detection limit of this assay with cultures of Salmonella Typhimurium ATCC 13311 was 10(1) colony-forming units (CFU)/mL. After 18-h enrichment, sigDE-based rt-RT-PCR could detect as low as 10(0) CFU/mL of Salmonella from egg broth and milk.

RNA-Based Detection Does not Accurately Enumerate Living Escherichia coli O157:H7 Cells on Plants

“…Therefore, unlike DNA-based detection, mRNA would only be limited to the viable and active cells within the population. Based on this assumption, reverse-transcriptase quantitative PCR (RT-qPCR) assays have been developed to detect a variety of foodborne pathogens such as enterohemorrhagic E. coli , Listeria monocytogenes, Salmonella , Vibrio , and Campylobacter species ( Klein and Juneja, 1997 ; Sheridan et al, 1998 ; Szabo and Mackey, 1999 ; McIngvale et al, 2002 ; Yaron and Matthews, 2002 ; de Wet et al, 2008 ; D’Souza et al, 2009 ; Miller et al, 2010 ; Techathuvanan et al, 2010 ; Kurakawa et al, 2012 ; Zhou et al, 2014 ). However, while some reports have concluded that mRNA disappears quickly after cell death ( McIngvale et al, 2002 ), other findings suggest that transcripts can persist for extended lengths of time (e.g., Sung et al, 2005 ; Xiao et al, 2012 ).…”

New bioluminescence pyrophosphate assay for high‐sensitivity detection of food‐borne pathogens

“…Although in several recent reports, some detection methods based on qPCR detected templates as low as 10–40 copy/μl or 10–100 CFU/ml (close to the sensitivity of the optimized bioluminescence pyrophosphate assay in this study), they all required quite expensive equipment and complex methods . However, in comparison, the assay proposed in this study costs less and is simpler and more convenient.…”

Emerging Biorecognition and Transduction Schemes for Rapid Detection of Pathogenic Bacteria in Food

“…For example, Lee and others () measured S . Typhimurium and Salmonella Enteritidis in beef and pork at levels as low as 10 3 CFU/mL in as few as 6 h. One of the most promising PCR developments involves combination of qPCR with a reverse transcriptase technique (or RT‐qPCR), which has been used as a method for distinguishing between viable and nonviable cells (Zhou and others ). Remarkably, Zhou and others () detected viable Salmonella at a detection limit of 10 1 CFU/mL in egg broth and milk using the Salmonella ‐specific sigDE operon as a viability indicator, together with IMS and solvent extraction.…”

“…Viability can be estimated by pretreating samples with reagents such as propidium monoazide (Li and others ) or monitoring the sigDE operon over time (Zhou and others ).…”

“…This advanced PCR technique is costly, but has one of the lowest LOD values in the literature for a culture‐independent technique associated with viability testing. This method has great promise, and use of viability indicators such as sigDE operon are under investigation by a number of labs to refine the method (Barnett and others ; Shao and others ; Ruff and others ; Garrido and others ; Garrido‐Maestu and others ; Li and others ; Vondrakova and others ; Zhou and others ; Meng and others ; Sebastião and others ). The drawback of this technique is the need for continuous monitoring of viability markers, complicating the assay, and adding analysis time and adding additional cost.…”