Octaplex PCR and fluorescence-based capillary electrophoresis for identification of human diarrheagenic Escherichia coli and Shigella spp.
A multiplex PCR assay, amplifying seven specific virulence genes and one internal control gene in a single reaction, was developed to identify the five main pathotypes of diarrheagenic Escherichia coli and Shigella spp. The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC). Each forward primer was labelled with a fluorochrome and the PCR products were separated by multicolour capillary electrophoresis on an ABI PRISM310 Genetic Analyzer (Applied Biosystems). If present, several gene variants of each virulence gene were identified. The internal control gene rrs, encoding 16S rRNA, was amplified in all 110 clinical strains analyzed. Virulence genes were demonstrated in 103 (94%) of these strains. In the majority of the cases (98/103, 95%), classification obtained by the novel multiplex PCR assay was in agreement with that previously determined by phenotypic assays combined with other molecular genetic approaches. Numerous multiplex PCR assays have been published, but only a few of them detect all five E. coli pathotypes within a single reaction, and none of them has used multicolour capillary electrophoresis to separate the PCR products. The octaplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the five main pathotypes of E. coli, and Shigella spp. This assay will replace the previous molecular genetic methods used in our laboratory and work as an important supplement to the more time-consuming phenotypic assays.
Brandal
Lin Thorstensen
LT
Division of Infectious Disease Control, Norwegian Institute of Public Health, Geitmyrsveien 75, P.O. Box 4404 Nydalen, Torshov, N-0403 Oslo, Norway. lith@fhi.no
Lindstedt
Bjørn-Arne
BA
Aas
Lena
L
Stavnes
Trine-Lise
TL
Lassen
Jørgen
J
Kapperud
Georg
G
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003967:Diarrhea / Q000382:microbiology*; D004405:Dysentery, Bacillary / Q000382:microbiology*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D022622:Shiga Toxin 1 / Q000737:chemistry / Q000235:genetics; D022641:Shiga Toxin 2 / Q000737:chemistry / Q000235:genetics; D012760:Shigella / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics; D014774:Virulence
D018829:Adhesins, Bacterial; C088603:AggR protein, E coli; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D015534:Trans-Activators; C507120:ipaH protein, E coli; C063500:ipaH protein, Shigella flexneri; C073819:eaeA protein, E coli