title: Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae genes of Escherichia coli O157:H7.
abstract: A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to rfbE and eae, respectively, were targeted for reverse transcription, amplification, and real-time detection. A single-step RT-PCR kit containing a mixture of reverse transcriptases converted mRNA into cDNA, which was subsequently amplified by Taq polymerase included in the same kit. The real-time detection of amplification products was achieved by incorporating rfbE(O157)- and eae(O157:H7)-specific TaqMan probes in rRT-MPCR. The ability of two sets of primers and probes for specific detection of rfbE(O157) and eae(O157:H7) was initially verified by screening RNA of eight E. coli serotypes possessing different O antigens and eae alleles. These two sets of primers and probes were also tested in a standard real-time PCR (rPCR) using DNA prepared from several E. coli and non-E. coli strains to verify that only rfbE(O157)- and eae(O157:H7)-specific sequences were amplified and detected. The rRT-MPCR was then evaluated for detecting low-level contamination of EHEC O157:H7 in feces. When RNA prepared from bovine feces, which were artificially seeded with EHEC O157:H7 cells and cultured for five hours, was tested in rRT-MPCR as low as 1cfu/g of feces could be detected. The detection range for the two genes in fecal cultures was 5.1 x 10(-1)-5.1 x 10(4) cfu/g of feces. Thus, the described procedure could be applied to rapid detection of very low levels of EHEC O157:H7 using total RNA as a template. Since the presence of rfbE(O157)- and eae(O157:H7)-specific mRNA is dependent on replicating cells, rRT-MPCR could provide important information about the viability of EHEC O157:H7 in feces.
authors.affiliation: Pre-Harvest Food Safety and Enteric Diseases Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, P O Box 70, Ames, IA 50010, USA. vsharma@nadc.ars.usda.gov
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Sharma
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis*; D000818:Animals; D002238:Carbohydrate Epimerases / Q000032:analysis*; D002417:Cattle / Q000382:microbiology*; D017931:DNA Primers / Q000737:chemistry; D019453:Escherichia coli O157 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis*; D005243:Feces / Q000737:chemistry / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012333:RNA, Messenger / Q000032:analysis; D000637:Transaminases / Q000032:analysis*
chemical_list: D018829:Adhesins, Bacterial; D017931:DNA Primers; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; C073819:eaeA protein, E coli; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase