Design "on-demand" of pathogen-specific qPCRs

 

If you need a qPCR to detect a gene or a microbial pathogen not found on the actual available list of GPS™ kits, please contact our experts.

GPS™ may design, produce, and validate, kits for real-time PCR on demand (“a la carta”), to detect any pathogen (or any genetic target), at several taxonomic levels: from the genus, species, or specific strains. 

Please send us the name of the species (or taxon, or gene) you need to detect and experts at GPS™ will perform a preliminary non-committed phylogenetic "in silico" analysis of available data. 30 years of experience on microbial phylogenetic diversity and taxonomy, together with demonstrated skills in design and validation, ensures the best possible real-time PCR assay following the strictest criteria. The design, development and validation of the new assay is free-of-charge, only small administrative costs will be charged at time of first order this new kit.The new GPS™ qPCR kit will be ready for shipment in 2-3 weeks from the date your order is activated

The obtained qPCR kit will be with the same characteristics as these actually provide by GPS™, in several formats: as TargetSpecies dtec-qPCR Test for 100 and 300 reactions; or as a MONODOSE format MONODOSE dtec-qPCR Test  for 24, 48, 96 reactions (DNA target) or for 60 and 120 reactions (RNA target).

  • The tests include highly specific oligonucleotide primers and dual-labelled probes, but also end-point PCR kits (only primers) may be developed to be use in conventional PCR or with nucleic acid intercalating dyes (not supplied).
  • The tests are suitable for ALL quantitative thermal cyclers available in the market. Our mastermix contains an optimized mixture compatible with both, capillary and plate based qPCR devices.
  • The tests include also DNA standard template with known copy number in a lyophilized format to build calibration curves or use as positive control.

GPS criteria on specific targets for microbial detection 

The design of real time PCR kits for detection of specific pathogens (genera, species, or strains) requires a broad experience and optimization work. At GPS™, we design and package real time PCR primers/probes for any target gene, all mixed and lyophilized in a single tube at optimal concentration for qPCR reactions. Send the name of the species for a preliminary free-of-charge phylogenetic "in silico" analysis of available data and we will produce and package ready-to-use the primers/probe mix for your specific research needs.

If you need a standard targeted DNA with precise copy number of template for calibration curves to be build, also suitable for positive extraction control, GPS™ may provide the synthesized and cloned amplicon in a lyophilized format.

The tests include highly specific oligonucleotide primers and dual-labeled probes, but also nucleic acid intercalating dyes may be used. They tests aresuitable for ALL PCR thermocyclersavailable in the market.

Specificity of targets for microbial detection

The sequence specificity of gen targets is crucial in the design of primers for species-specific detection. The target sequence should be conserved for all strains of a single species (inclusivity) but also should show enough nucleotide variation or mismatches to these homologous sequences from all the other, related or not, species and genera (exclusivity). Above atributes highly relative as they depend of:

  • the species: not all species evolve at the same rate
  • evolving nature of the sequences: not all show the same evolution mode, conservation
  • and more difficult, the diversity/data available at public databases

Consequently, very often, most available primers and probes published and available in commercial kits fail to detect some strains of the target species, or give false positives in the presence of phylogenetically related taxa.

GPS™ expertise in microbial evolution provides robust frames for this purposes. Our team uses phylogenetic criteria to select the best possible target for species-specificity guarantied.

Sequence design of primers and probes Universally known and requested primer/probe attributes are under control by personal dedication of our experts and using the best software available for design of qPCR primers and probes. An optimal design for real time PCR includes the following characteristics: 

  • Minimise primer dimers
  • Minimise primer hairpins
  • Optimal Tm
  • Optimal G+C content
  • 3'-end GC clamp Short amplicons
  • No template secondary structure
  • Probe melting point
  • Probe length
  • Probe G/C content

 

GPS know-how ensures robust designs with high degree of specificity

*GPS primers and probes are sold for research use only. 

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