I have been isolating 120 E. coli from rectal swabs of cattle to confirm whether there is any O157:H7. I have cultured them onto a selective media - Sorbitol MacConkey agar with Cefixime Tellurite supplement (SMAC). As per the recommendation, E. coli O157:H7 organisms produces colorless colonies but other E. coli produce pink colonies on the media. I got 4 isolates among the 120 isolates producing colorless colonies on SMAC. I did a PCR of these isolates with rfbO157F: aag att gcg ctg aag cct ttg, rfbO157R: cat tgg cat cgt gtg gac - primers but did not get any result. What is the reason behind this?
On CT-SMAC agar colonies appear clear due to their inability to ferment sorbitol. However, colourless colonies similar to O157 may also grow on this agar. It is advisable to use two Media ie Chromagar in addition to CT-SMAC. Colonies of O157 appear mauve in colour on Chromagar. Colonies should also be tested for the presence of the somatic O157 antigen by latex agglutination before moving to PCR.
I totally agree with the previous comment. You also can add other steps in your protocol like concentration of E. coli O157 by immunomagnetic separation with Dynabeads anti-E. coli O157. However it will increase the final cost of your protocol.
@Mohamed Ahmed
I used a positive control. I also screened. the isolate for Stx1 and Stx2 where they were positive for both Stx1 and Stx2. So, I am sure about the purity of the extracted DNA. The organisms were isolated from the rectal swab of cattle.
Do you think I should use other sets of primer? There are some other primer sequences used by most of the researchers. I am thinking to use the primers along with the chromagar.
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