Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment.
OBJECTIVE
To develop a real-time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin-producing E. coli (STEC) serotypes classified in seropathotype C.


RESULTS
The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan minor groove binder probe specific for fliC-H21 were designed and used in a 5'-nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC-H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was detected after overnight enrichment.


CONCLUSIONS
The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef.


CONCLUSIONS
The real-time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.
Auvray
F
F
Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et les Procédés agroalimentaires (AFSSA-LERQAP), Maisons-Alfort, France. f.auvray@afssa.fr
Lecureuil
C
C
Taché
J
J
Perelle
S
S
Fach
P
P
D000818:Animals; D001483:Base Sequence; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005408:Flagellin; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics* / Q000378:metabolism*
D029968:Escherichia coli Proteins; C516095:FliC protein, E coli; D005408:Flagellin