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REAL‐TIME REVERSE TRANSCRIPTION PCR DETECTION OF VIABLE SHIGA TOXIN‐PRODUCING ESCHERICHIA COLI O157:H7 IN FOOD
Author(s) -
DI R.,
TUMER N.E.
Publication year - 2010
Publication title -
journal of food safety
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 43
eISSN - 1745-4565
pISSN - 0149-6085
DOI - 10.1111/j.1745-4565.2009.00189.x
Subject(s) - escherichia coli , microbiology and biotechnology , biology , trizol , reverse transcription polymerase chain reaction , shiga toxin , food microbiology , enumeration , polymerase chain reaction , reverse transcriptase , tryptic soy broth , virulence , bacteria , rna , rna extraction , gene , messenger rna , biochemistry , genetics , mathematics , combinatorics
Shiga toxin (Stx)‐producing Escherichia coli (STEC), particularly E. coli O157:H7, have been associated with food‐related outbreaks and have become a global health concern. Detection of low numbers of viable STEC in food is undoubtedly challenging. The present study demonstrated that 7 × 10 2 to 7 × 10 3 cfu E. coli O157:H7 cells in 50 mL of inoculated food samples including apple juice, lettuce and ground beef could be concentrated through a two‐step filtration technique. Bacterial RNA was effectively isolated from the concentrated E. coli O157:H7 cells with the combination of Trizol Max Bacterial RNA Isolation Enhancement Reagent and TriReagent®. Real‐time reverse transcription polymerase chain reaction with seven sets of primers targeting virulence and serotype genes of E. coli O157:H7 specifically detected the bacterial RNA representing 1 × 10 2 to 1 × 10 3 cfu viable bactersial cells. The total detection time from sampling to measurement was approximately 4 h. This study provides a rapid and specific detection method for viable STEC in food.PRACTICAL APPLICATIONS Rapid detection of viable STEC including Escherichia coli O157:H7 cells in food has been difficult, especially when present in low numbers. This study has shown that 7 × 10 2 to 7 × 10 3 cfu E. coli O157:H7 in 50 mL of inoculated food samples, i.e., 14–140 cfu/mL bacterial cells, could be readily concentrated and the bacterial RNA could be detected by real‐time reverse transcription polymerase chain reaction (PCR) in a relatively short time. Use of seven primer sets makes this method very specific for real‐time PCR detection of E. coli O157:H7.