Conventional + real-time (TaqMan) primer/probe design for species screening (class-level) and single-species identification panels — singleplex or multiplex. Built for JB Scientific. Not a clinical/regulatory diagnostic device; every assay must be wet-lab validated before use.
Starts empty — add any target below, or click "Load 45-species reference list" to bring in the species from your originally-uploaded list (river/marine fish, elasmobranchs, crustaceans, molluscs, livestock & poultry). Not limited to that list either way. Each target is auto-mapped to a broad screening class (used by the class-level screening panel in Tab 5).
Type a common trade name — e.g. "chicken", "sting ray", "beef" — a scientific (binomial) name, or a genus/family name, and this looks it up against a small curated lookup table built for this tool's own meat/seafood-adulteration domain (Pakistani/South Asian market names + the 45-species reference list) — it is not a general taxonomic database (not GBIF/ITIS/WoRMS/NCBI Taxonomy). Coverage: common terrestrial meat/poultry species (chicken, beef/cattle, buffalo, goat, mutton/sheep, pork, donkey, horse, mule, camel, rabbit, duck, turkey, + the screening/contamination species from the 45-species list) and seafood/fish groups with genuine multi-option ambiguity (ray/stingray, shark, snapper, grouper, pomfret, mackerel, tuna, sardine, catfish, pangasius, rohu/catla/mrigal carps, shrimp/prawn, crab, squid, salmon). A name not found here is not an error — it just means manual entry below, still with a full choice of rank (species/genus/family/class).
Use this directly for anything not in the curated lookup above, or to fully control every field yourself.
Download the template, fill it in with any spreadsheet app, and upload it here. Every row is matched through the exact same curated-lookup logic as the single-add box above — ambiguous rows are shown for you to resolve, unmatched rows are shown as manual, and nothing is added to your target list until you review and commit.
| # | Class | Rank | Category | Local name | Common name | Scientific name | Sequences on file |
|---|
Automatic, for every ticked target at once — no need to pull sequences one by one. Tries each configured source (NCBI, BOLD, and ENA — a third, independent database, since BOLD is often unreachable from a plain browser) in turn for every target and marker below, and saves whatever comes back. Anything that fails live gets flagged so you can fill the gap with the manual link/paste tools further down. Re-running skips targets that already have a saved sequence for that marker.
Species-ID and adulteration assays are almost always built on mitochondrial barcode markers — COI ("Folmer region"), cytochrome b (cytb), or 16S/12S rRNA — because they're multi-copy per cell (works on degraded/cooked/processed samples) and have deep public reference coverage. Use this for one target at a time — to patch a gap the bulk fetch above couldn't fill, or to pull a marker not covered by the bulk defaults.
Paste one FASTA record (header line starting with > is optional). This becomes available in Tab 4 for de novo design (Method 3) and consensus alignment (Method 2), and in Tab 2's Auto-Design as an already-saved sequence for that marker.
Give it a target list, a PCR type, and a plexing mode — it pulls two independent reference sequences per target from two different sources, designs a primer set from each (a probe too, for real-time), cross-checks them in-silico, draws the binding sites to scale, and — if multiplex — pools everything and works out cycling conditions. Uses the parameters set below (length/Tm/GC/homopolymer/clamp — collapsed by default, shared with Tab 4's manual methods); amplicon-size range is auto-tightened for real-time unless you've already set something narrower there.
Shared by all three sources above. Real-time (TaqMan) amplicons are usually short (60–150 bp); conventional gel-based amplicons — especially for a multiplex species-ID gel — are usually chosen longer and deliberately size-staggered (150–800 bp) so species can be told apart by band size. Real-time runs auto-tighten the amplicon-size range unless you narrow it below yourself here first.
These two feed Auto-Design's automatic pooling stage and saved-panel SOPs. Tab 5's manual multiplex screen has its own copies of the same controls (so you can re-screen an already-built pool without re-running design) — change either independently.
Classic TaqMan design rules applied as filters: no G at the 5′ end (quenches reporter fluorescence), fewer G's than C's preferred, avoid runs of ≥4 identical bases, probe sits fully inside the amplicon (not overlapping either primer).
Each stage below updates live as the run progresses and stays open for inspection afterward — click any stage to expand its detail. This is one reasonable pipeline shape for this workflow, not a fixed standard; stages that don't apply to a given run (e.g. HRM design when targets aren't closely related) are marked not-applicable rather than skipped silently.
Shared with Tab 2 · Auto-Design (same fields, same values — sensible defaults are already applied, so opening this is optional). Collapsed by default; any change here applies there too.
Three independent routes to primers/probes, as requested — use any or all and compare:
A small, hand-curated seed set of real, published primers/probes (universal barcoding sets + a few species-specific real-time assays for major adulterants). Every entry below carries its literature citation — nothing here is invented. This is a starting seed, not a complete panel: most of the 45 uploaded species do not yet have a published species-specific assay in this list, and need Method 2 or 3.
| Name | Scope | Gene | Type | Sequence (5′→3′) | Len | Tm | GC% | Amplicon | Citation |
|---|
How it works: paste 2 or more pre-aligned homologous sequences (same marker, same length — align them first below, in MUSCLE/Clustal/MEGA, or export already-aligned from BOLD). Mark which one is your target species. The tool scans column-by-column for (a) fully conserved stretches long enough to serve as universal/flanking primer sites, and (b) diagnostic positions where the target differs from every other sequence pasted — good centers for a species-specific probe or an allele-specific 3′-mismatch primer.
Pick any combination of sequences already saved against your targets (Tab 3 · Sequence Retrieval — including anything pulled by the bulk-fetch card or Auto-Design), and/or paste extra ones, then align them with this tool's own built-in aligner. It's a basic pairwise-to-a-reference (center-star) global aligner — good enough to equalize lengths and line up a barcode region for closely related species, but it is not a substitute for MUSCLE/Clustal/MAFFT on a divergent or hard alignment; for anything that looks wrong, align externally and paste the result into the box below instead.
How it works: paste (or pull from Tab 2) a single target-species sequence. The tool slides candidate forward/reverse primer windows across it (Primer3-style scan-and-score against the parameters above), pairs them into amplicons in your size range, and — if probe design is on — picks a TaqMan probe inside each amplicon.
Every primer/probe you "add to pool" from Tab 2 (Auto-Design) or Tab 4 (curated/consensus/de novo) lands here. Build your screening panel and single-species ID panel from this pool, then run compatibility screening.
| Target / scope | Method | Gene | Name | Type | Sequence | Tm | Amplicon |
|---|
Coarse first-pass screen: "is this plant, bird, land-mammal, or aquatic material?" Built from curated universal primer sets per class (Method 1). Select the classes present in your target list and add their universal primers straight into the pool above.
Select which pool entries go in the same reaction/gel run. The tool checks: nearest-neighbor Tm spread (all primers should anneal within one program), 3′-end cross-dimer risk (heuristic — see disclosures), and, for conventional multiplex, whether amplicon sizes are far enough apart to resolve as separate gel bands. Incompatible items are automatically split into the minimum number of pools via graph colouring.
Everything currently in the design pool (Tab 5).
Project state (targets, saved sequences, pool) also autosaves to this browser's local storage, so reopening this file on the same computer picks up where you left off.
Save a named snapshot of the current design pool (from Auto-Design, curated Method 1, or manual Method 2/3 — whatever is currently in Tab 5's pool) with its own downloadable SOP report, so you can build up a library of panels over time and come back to any of them later — without re-designing from scratch. Saved panels persist in this browser's local storage alongside the rest of the project.