Hi

In first you Bacteria isolates cultures were grow on MRS -Sorbitol agar. This media to help you for numbers reduce of isolates and selective media for E.coli O157. 

Alaa, while you are sort of correct for 95% of O157 strains I would suggest Jawa still runs PCR. This for two reasons:

1/. Sorbitol will only detect 0157 isolates and only 95% of O157 isolates will be positive on sorbitol, hence some O175 will be missed

2/. Majority of stx strains is NON 0157 and thus most stx strains will be missed on sorbitol plates. There is better plates but still they will not detect all Stx positives.

So PCR is best solution. We also run multiplexes, but problem with multiplex is that they are not easy to transfer to other equipment, taq, buffer, etc. etc. so one would need to perform extensive validations. If there is not too many strains (

hi

I used 58 as optimum annealing for four different strains of E.coli.

Hi,

In my lab we used multiplex PCR for detecting simultaneouslu eae, stx1 and stx2 genes by PCR. If you interested please read the next paper:1.      Stanilova S, Rusenova N, Petrova D, Stoyanchev T, Polymerase chain reaction assay for detection of enteropathogenic Eshherihia coli strains in meat. Trakia Journal of Sciences, 2011, 9, 51-57.

I have been working on pathogenic E.coli for the last 5 years and i have not had any problem with an annealing temp of 59 for 1min 30sec

thank you all for the answers so far.

I send my lab conditions:

eaeA  F:5´-GAC CCG GCA CAA GCA TAA GC-3´

R:5´-CCA CCT GCA GCA ACA AGA GG-3´

 95°C 44’’

50°C 45’’

72°C 45’’

Stx1 F:5´-CTG GAT TTA ATG TCG CAT AGT G-3´

R:5´-AGA ACG CCC ACT GAG ATC ATC-3´

95°C 45’’

50°C 45’’

72°C 45’’

Stx2 F:5´-GGC ACT GTC TGA AAC TGC TCC-3´

R:5´-TCG CCA GTT ATC TGA CAT TCT G-3

95°C 45’’

50°C 45’’

72°C 45’’

I am currently using Dream Taq  with the following conditions 95  5min

94 1min 59 1min 30sec 72 10 min for 25 cycles and finally 72 10 min. You can give this a shot

While you evaluate the information provided, I strongly recommend you take note of Johannes Kusters's suggestion on multiplexing. It can be difficult to replicate the multiplexing technique at times.

All the best