Question: Design Pcr Primers To Uniquely Identify A Bacterial Strain
gravatar for Eric Normandeau
7.0 years ago by
Quebec, Canada
Eric Normandeau10k wrote:

I am trying to help a group develop a pair of PCR primers that would permit the accurate detection of one specific bacteria strain. The goal is, using a simple PCR kit, to be able to determine whether the studied strain is in the mix.

I am not used to work with bacteria or metagenomes, so I have a few questions regarding the feasibility and best approach to design these primers.

Some options that we are considering:

Option A:

  • design primers in a highly variable region of a gene that is often used for bacteria identification (eg: 16S, GroES, GroEL, rpoD, GCAT, gyrB, cpn60, rpoB)

Option B:

  • Get all the bacterial genome data that is available
  • Identify kmers that are found only once in our strain and never in all the bacterial data, including other strains of the same species
  • Look for pairs of those unique kmers that are separated by 100-800 pb in the target strain's genome to design primers
  • Do some sequencing of the targeted strain and similar strains to confirm that the primers are unique to the targeted strain
  • Test in silico amplification in all other genomes (bacterial AND eukaryotes)
  • Test it in the lab (positive and negative controls...)

Of course, we would test in the lab for false positive/negative results.

My questions are:

  • Is using a single PCR likely to be powerful enough to detect the presence of one specific strain with low false positives/negatives?
  • What approach would you use to design PCR primers that would be specific to one strain?
bacteria pcr • 7.4k views
ADD COMMENTlink modified 6.9 years ago by Biostar ♦♦ 20 • written 7.0 years ago by Eric Normandeau10k

Did you search a medical routine techniques for identification pathogenic strains? I think, this is the same problem

ADD REPLYlink written 7.0 years ago by irinagaranina2410

No, I did not think of that and I will try. Would you have examples of publications?

ADD REPLYlink written 7.0 years ago by Eric Normandeau10k

Here are few papers that talks about strain specific primer design.

All these publication seem to design 4-5 primer sets (ranging from housekeeping to specific genes) and then test it in vitro to chose the best one.

ADD REPLYlink written 6.9 years ago by microbeatic80

Thank you, I'll read those.

ADD REPLYlink written 6.9 years ago by Eric Normandeau10k
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