Abstract: Diarrheagenic E. coli strains contribute to water related diseases
in urban and rural environment in developing and developed world. E. coli
pathotype and pathogenicity varies due to complex multifactorial mechanism
involving a large number of virulence factors. Rapid assessment of the
virulence pattern of E. coli isolates is possible by Real-Time PCR probes like
TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E.
coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were
retrieved from NCBI's GenBank database. The alignment of the multiple sequences
and analysis of conserved sequences was carried out using ClustalW and BLAST
programs. The primers and Taqmen probes were designed using Beacon Designer
software version 2.1 for two multiplexed PCR assays. In silico PCR simulation
of these assays showed PCR products for stx2 (248 bp) stx1 (102 bp), lacZ
(228bp) and lamB (86 bp) in multiplex #1 and eae (200 bp), chuA (147 bp), hlyA
(141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR
amplification products and probes can be used to identify and confirm presence
of O157:H7/H7-, O157:H43/45 and O26:H^{-}/H11 serotypes. In
conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan
probes designed and validated in silico will be helpful in management of water
quality and outbreaks, by improving specificity and minimizing time needed for
in vitro verification work.
Keywords: E. coli O157, TaqMan probe, polymerase chain reaction
