I developed a multiplex PCR assay for STEC detection targeting eae (384 bp), stx1 (180 bp), stx2 (255 bp), O111 (146 bp), O103 (98 bp), O45 (65 bp), O157 (259 bp), O26 (135 bp), O145 (132 bp), and O121 (98 bp). When I combined all of the positive controls in one reaction, only the stx and eae bands amplified, while all serogroup targets failed to amplify. I therefore divided the assay into two panels, which worked properly. Has anyone experienced a similar issue? Could this be due to primer competition or multiplex PCR optimization problems?
Hi everyone! I’m a master’s student in microbiology working on the detection of Shiga toxin-producing E. coli (STEC) in meat samples using culture methods and PCR.
I’m still new to laboratory work and I recently faced a problem with my PCR. My positive control did not amplify, and all of my samples were negative as well. I used mTSB enrichment before PCR.
What could be the possible reasons for a positive control failing to amplify? Could it be an issue with the DNA, primers, PCR conditions, or something else? I would really appreciate any advice or troubleshooting tips.