title: Detection of live Escherichia coli O157:H7 cells by PMA-qPCR.
abstract: A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.
authors: Center for Food Safety and Applied Nutrition, Division of Molecular Biology, Food and Drug Administration.
authors: Baoguang
authors: B
authors: Li
authors: Zonglin
authors: Z
authors: Hu
authors: Christopher A
authors: CA
authors: Elkins
mesh_terms: D001386:Azides / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000032:analysis / Q000187:drug effects / Q000235:genetics; D004783:Environmental Microbiology; D019453:Escherichia coli O157 / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D005516:Food Microbiology / Q000379:methods; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium