Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.
The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.
Miyamoto
Takahisa
T
Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. tmiyamot@agr.kyushu-u.ac.jp
Ichioka
Natsuko
N
Sasaki
Chie
C
Kobayashi
Hiroshi
H
Honjoh
Ken-ichi
K
Iio
Masayoshi
M
Hatano
Shoji
S
D000818:Animals; D001483:Base Sequence; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology*; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D017931:DNA Primers; D004269:DNA, Bacterial