Basically, the primers are same for all types of PCR as you will amplify same target DNA. But for realtime PCR you should additionally use Taqman or SYBR green probe and your amplicons must be smaller than conventional PCR due to higher sensitivity of realtime. In turn to multiplex PCR, as you will use different primers for different bacteria but it is very important to have their Tm or annealing temperatures very closer or similar.

You can use online tool for desining your priners considering above things.

Best of luck.

For convectional PCR, the amplicon size can be of any size but coming to RT PCR, the amplicon size should be less than or around 100bp size to get the accurate results, for that you need to design primer accordingly, suppose if your amplicon for convectional PCR is around 1000 bp size than you need to redesign a new primer for RT PCR from the same amplicon sequence such that your amplicon size should be around 100 bp.

In multiplex PCR, definitely you have to use more than 2 sets of primers for detecting the same organism as their target sites are different in same genomic DNA

so, as per the requirement you need to use different primers accordingly to get accurate results.

• In terms of primer design you can substitute RT PCR primers for more quantitative analysis like sybr green qPCR
• Thus, the same rules in terms of optimal primer design apply
• The only caveat I would make is that your Tmfor such primers for sybr green qPCR should be 60C to 65C; With conventional 3 step PCR and RT PCR it can be lower as you have a separate annealing temp, where in you can go down to 50C and then a 3rd iteration @ 72C for extension
• For sybr green qPCR like 2 step RT PCR your annealing and extension are combined so you cannot really drop below 60C as the extnsion by the polymerase becomes too inefficient, and primer efficincy is paramount for qPCR
• In addition, specifically for qPCR and not RT PCR you should not end your primer in a T residue and as per standard primer design TT or AA are definitely contar indicated as that yould result in inefficient priming
• Thus, as per standard primer design with this one caveat you can end primer in CA GA but ideally CC; GC; GG
• As already mentioned because primer efficincy must be > 85% you need to keep your amplicon at 100bp
• Regarding efficiency, you also need to verify that it is above 85% by performing a serial dilution of your cDNA and performing a standard curve using a standard curve program in your qPCR software
• Only then do you perform actual qPCR to enumerate gene expression

Some quantitative PCR methods (e.g. Scorpions) use specially designed labelled primers, but most methods use ordinary, unlabelled oligos. The most usual way of providing a signal for quantification is to use a 5' hydrolysis (TaqMan) probe. This also provides extra specificity.

Do check for cross-reaction with non-pathogenic Listeria strains like L. innocua. They're a lot more common and abundant that L. monocytogenes.

Thank you for your generous answers

Hi

The answer is both Yes and No. You can select one set of primer and it can be used for conventional and real time PCR or any other formats. However, real time PCR additionally requires probes for discrimination and quantification. I agree with the suggestion provided by Andrew Jenkins. You can use these methodologies for multiplex also. It is better to avoid SYBR methodology for quantification due to the nature of non-specific binding with any dsDNA.

Sometimes, the answer is No. The amplicon size is often larger in conventional PCR whereas real time PCR provides better and superior results often at ampliocn size less than 200 base (ideal is less than 150 bp). In this case, it is not possible to convert conventional into real time primers and probes.