title: Development of a sigDE-based real-time reverse-transcriptase PCR for the detection of viable Salmonella 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.
authors.affiliation: 1 MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology, and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University , Shanghai, China .
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Jielin
authors.initials: J
authors.lastname: Yang
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Daixin
authors.initials: D
authors.lastname: Liu
authors.forename: Chengang
authors.initials: C
authors.lastname: Yuan
authors.forename: Yuping
authors.initials: Y
authors.lastname: He
authors.forename: Liangwen
authors.initials: L
authors.lastname: Pan
authors.forename: Xianming
authors.initials: X
authors.lastname: Shi
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial; D055028:Comparative Genomic Hybridization; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004531:Eggs / Q000382:microbiology; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000175:diagnosis / Q000382:microbiology; D057230:Limit of Detection; D008892:Milk / Q000382:microbiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019779:Salmonella enterica / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D011938:Rec A Recombinases