Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction

primers; i.e., theycontain intentionally introduced sequence ambiguities toovercome minorsequence variations within different SLTgenes. Indirect gelhybridization withgenomic DNA,both primers recognized SLT-IandSLT-ll DNA sequences. Amplified sequences oftarget DNA obtained by polymerase chainreaction werevisualized after gelelectrophoresis byethidium bromidestaining, and definitive identification oftheamplification product asanSLTgenesegment wasachieved byhybridization to SLT-I- andSLT-H-specific 20-base oligonucleotide probes complementary toa portion oftheamplified sequences butnottotheprimers. Thedetecting oligonucleotide probes shared only30%basehomology and wereshowntorecognize specifically SLT-IorSLT-II sequences within genomic DNA.Morever, they wereused todistinguish whether theamplified sequence originated fromSLT-IorSLT-II genes. ThePCRsystem with theprimers described hereisapowerful technique toamplify SLTsequences inE.coli strains thatproduce serologically distinct SLTsandwill facilitate identification ofthese pathogens, particularly amongamultitude ofnonpathogenic E.coli strains.

[1]  J. Brown,et al.  Determination by DNA hybridization of Shiga-like-toxin-producing Escherichia coli in children with diarrhea in Thailand , 1989, Journal of clinical microbiology.

[2]  H. Karch,et al.  Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs. , 1989, FEMS microbiology letters.

[3]  M. Karmali,et al.  Infection by verocytotoxin-producing Escherichia coli , 1989, Clinical Microbiology Reviews.

[4]  T. Gingeras,et al.  Nucleic AcidAmplification InVitro: Detection ofSequences with LowCopyNumbersandApplication toDiagnosis ofHuman Immunodeficiency Virus Type1Infection , 1989 .

[5]  J. H. Green,et al.  Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies , 1988, Infection and immunity.

[6]  R. Neill,et al.  DNA probes for Shiga-like toxins I and II and for toxin-converting bacteriophages , 1988, Journal of clinical microbiology.

[7]  Andjohnj . Mekalanos.  Nucleotide sequence oftheShiga-like toxin genes ofEscherichia coli , 1987 .

[8]  J. Wells,et al.  Production of Shiga-like toxin by Escherichia coli. , 1986, The Journal of infectious diseases.

[9]  R. Holmes,et al.  Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities , 1986, Infection and immunity.

[10]  H. Lior,et al.  The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. , 1985, The Journal of infectious diseases.