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  • Multiplex PCR detection of Shiga toxin genes
    Cheryl L. Tarr, Sean Reid, and Thomas S. Whittam

    The multiplex amplification reaction was performed in a total of 25 uL with 1.5 units of AmpliTaq Gold in the AmpliTaq Gold buffer supplied by the manufacturer (Applied Biosystems). Final concentration of reaction components were as follows: 0.2 uM each primer; 0.2 mM each dNTP; and 2 mM MgCl2. A total of six primers were used: mdh p9 and mdh p10, which produce a 201 bp product that serves as an internal positive control; 2A.506F and 2A.848R, which yield a 383 bp fragment of the Stx 2 gene; and 1A.251F and 1A.832R, which amplify a 624 bp fragment of Stx 1. Approximately 100 ng of template DNA was added to the reaction. The thermal profile, which was preceded by a 10 minute incubation at 94 degrees C, was run for 35 cycles with the following parameters: 92 C, 40 sec; 60 C, 1 min; 72 C, 2 min.

    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 p9	  cta acc cgg tta aca cca cag t
    mdh p10   ggc aga atg gta aca cca gag t
    Stx primers
    1A.251F	  ggg ata gat cca gag gaa gg
    1A.832R	  ccg gac aca tag aag gaa act c
    2A.506F	  ctg gcg tta atg gag ttc ag
    2A.848R	  cct gtc gcc agt tat ctg ac

    Primers for the Shiga toxin genes are numbered according to the following system: the first number refers to the Stx locus (Stx 1 or Stx 2); the letter refers to the subunit in which the primer is located (A or B); the next number refers to the position of the 3' base of the primer in the gene. Forward primers (F) match the coding (sense) strand.

    Recipe per 25 uL reaction:
    DNA (100 ng/uL)           1.0  uL
    10 X buffer               2.5  uL
    dNTP's (2 mM each)        2.5  uL
    MgCl2 (25 mM)		  2.0  uL
    mdh p9 (10 uM)		  0.5  uL
    mdh p10 (10 uM)		  0.5  uL
    1A.251F (10 uM)		  0.5  uL
    1A.832R (10 uM)		  0.5  uL
    2A.506F (10 uM)		  0.5  uL
    2A.848R (10 uM)		  0.5  uL
    Amplitaq Gold (5 U/uL)    0.15 uL
    ddH2O                    13.85 uL
    Operated by Shannon D. Manning at Michigan State University