Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker.
The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.
Division of Molecular Biology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, Maryland, USA. baoguang.li@fda.hhs.gov
Baoguang
B
Li
Jin-Qiang
JQ
Chen
D001386:Azides; D017931:DNA Primers / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics* / Q000254:growth & development; D059022:Food Safety / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D011419:Propidium / Q000031:analogs & derivatives; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013234:Stem Cells
D001386:Azides; D017931:DNA Primers; D005819:Genetic Markers; C533957:propidium monoazide; D011419:Propidium