Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins.
This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes. The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC). Both generic methods detected all stx genes. In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f. Four of the subtyping protocols identified stx genes in all of the field isolates. The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested. The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers. One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes. This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction.
Ziebell
Kim A
KA
Health Canada, Laboratory for Foodborne Zoonoses, Guelph, ON. kim_ziebell@hc-sc.gc.ca
Read
Susan C
SC
Johnson
Roger P
RP
Gyles
Carlton L
CL
D000818:Animals; D002522:Chlorocebus aethiops; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D022622:Shiga Toxin 1 / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D022641:Shiga Toxin 2 / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D014709:Vero Cells
D004269:DNA, Bacterial; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2