Forget PCR. Forget pathogenic bacteria and P2. Your focus now is on just two pairs of sequences: Primer #1R and its matching E.coli sequence and Primer #2R and its matching sequence. You are going to ask BLAST to compare each pair, just the 20 nucleotides with no chance for confusion by extraneous E.coli or P2 sequences. You can see with your own eyes that the two pairs are equally good matches. Unless the BLAST has gone crazy, it will agree.

First you try Primer #1R and it's match [you can copy and paste from the inset two slides ago].

That is very good. Now you try Primer #2R and its match, very carefully -- no sudden motions --- so as not to give the program any cause for...

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