Design Pcr Primers To Uniquely Identify A Bacterial Strain
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7.5 years ago

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 • 7.7k views
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Did you search a medical routine techniques for identification pathogenic strains? I think, this is the same problem

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

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

http://www.sciencedirect.com/science/article/pii/S1075996409000729 http://link.springer.com/article/10.1007/s10658-013-0225-4

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.

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Thank you, I'll read those.

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