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  • Multiplex PCR detection of intimin and Shiga toxin genes
    David W. Lacher, Seth T. Walk, Lukas M. Wick, and Thomas S. Whittam
    February 2004

    The multiplex amplification reaction was performed in a total of 25 µL with 1.5 units of AmpliTaq Gold in the AmpliTaq buffer II supplied by the manufacturer (Applied Biosystems). Final concentration of reaction components were as follows: 0.2 µM each primer; 0.2 mM each dNTP; and 2 mM MgCl2. A total of eight primers were used: mdh-F41 and mdh-R875, which produce a 835 bp product that serves as an internal positive control; eae-F626 and eae-R1166 which generate a 541 bp fragment of the central conserved domain of eae; stx1A-F122 and stx1A-R813, which yield a 691 bp fragment of stx1A; and stx2A-F481 and stx2A-R864, which amplify a 384 bp fragment of stx2A.
    Approximately 50 ng of template DNA was added to the reaction. The thermal profile, which was preceded by a 10 minute incubation at 94 degree C, was run for 35 cycles with the following parameters: 92 degree C, 1 min; 55 degree C, 1 min; 72 degree C, 30 sec. A final stage of 72 degree C for 5 min. was used for completion of any partially extended product. Amplification products were electrophoresed in 1.5 % agarose gels buffered in 1X TAE and visualized under UV illumination after staining with ethidium bromide.
    Primer sequences (5' to 3'):
    mdh-F41        AGG CGC TTG CAC TAC TGT TA
    mdh-R875       AGC GCG TTC TGT TCA AAT G
    eae-F626       ATT ATG GAA CGG CAG AGG TTA AT
    eae-R1166      ATC CCC ATC GTC ACC AGA GG
    stx1A-F122     CAT TCG CTC TGC AAT AGG TA
    stx1A-R813     AAC TCG CGA TGC ATG ATG A
    stx2A-F481     TAT CTG GCG TTA ATG GAG TT
    stx2A-R864     CCT GTC GCC AST TAT CTG AC
    	
    Recipe per 25 µL reaction:
    DNA (50 ng/µL)                    1.00 µL
    10X buffer                        2.50 µL
    dNTP's (2 mM each)                2.50 µL
    MgCl2 (25 mM)                     2.00 µL
    eae-F626 (10 µM)                  0.50 µL
    eae-R1166 (10 µM)                 0.50 µL
    mdh-F41 (10 µM)                   0.50 µL
    mdh-R875 (10 µM)                  0.50 µL
    stx1A-F122 (10 µM)                0.50 µL
    stx1A-R813 (10 µM)                0.50 µL
    stx2A-F481 (10 µM)                0.50 µL
    stx2A-R864 (10 µM)                0.50 µL
    Amplitaq Gold (5 U/µL)            0.15 µL
    ddH2O                            12.70 µL
    	
    Environmental E. coli optimization.The above protocol was used to screen both clinical isolates from disease outbreaks and environmental isolates from freshwater lakes and streams. Band intensities were lower for the environmental isolates and certain conditions of the protocol were optimized for these strains. These following changes were used to optimize the reactions for environmental strains. The thermal profile was run for 40 cycles instead of the 35 cycles used above.
    eae-F626 (5 µM)                     0.50 µL
    eae-R1166 (5 µM)                    0.50 µL
    mdh-F41 (10 µM)                     0.75 µL
    mdh-R875 (10 µM)                    0.75 µL
    stx2A-F481 (5 µM)                   0.50 µL
    stx2A-R864 (5 µM)                   0.50 µL
    	
    Example gel of multiplex PCR designed to produce amplicons for mdh (835 bp), stx1 (691 bp), stx2 (384 bp), and eae (541 bp). E. coli mdh primers are used as a control for a positive PCR reaction in each multiplex reaction. Lane 2: E2348/69, eae+ mdh+; Lane 3: K12, mdh+; Lane 4: Sakai, mdh+, eae+, stx1+, stx2+; Lanes 4-7, various environmental isolates; Lane 1 and 12 kb plus DNA ladder.
    Contact stec@cvm.msu.edu
    Operated by Shannon D. Manning at Michigan State University