In my experiment, i have used a specific primer for identification of E.coli o157 however, the positive control samples showed good results on gel electrophoresis but the random collected samples showed no results. the collected samples showed pink colonies when cultured on specific chromogenic E.coli agar the same as positive control.
The most commonly used primer for the identification of E. coli O157 using PCR is the O157 primers. These primers target a specific region in the O157 specific gene, which is unique to E. coli O157. The sequence of the primers is 5’-TCCTGTGCGATGAAGCTGC-3’ (forward primer) and 5’-CAGGTACTTCTTCCAACAC-3’ (reverse primer).
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It is possible that the specific primer you used may not be suitable for detecting all strains of E. coli O157, or that the sensitivity of the PCR reaction may not be sufficient to detect low levels of the target DNA in the samples. In order to select a more common primer for E. coli O157, you could search the scientific literature and databases to identify primers that have been used successfully in previous studies.
One approach could be to search for published papers or protocols that describe the detection of E. coli O157 using PCR and identify the primers that were used. You could also search for primers that have been designed specifically for the detection of E. coli O157 in commercial PCR kits. Once you have identified candidate primers, you could compare their sequences to ensure that they are specific for E. coli O157 and not other closely related bacteria.
It is also possible that other factors, such as the DNA extraction method or PCR conditions, may be affecting the sensitivity of the assay. You may want to optimize your PCR conditions by adjusting the annealing temperature, optimizing the PCR cycling conditions, or testing different DNA extraction methods to improve the sensitivity of your assay.
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