qpcr-primer-designer

为 RT-qPCR 设计引物和 TaqMan 探针，支持跨外显子、3' 端错配校验及同源映射硬核模式。

Security Scan

The skill's code, instructions, and resource access are consistent with an RT-qPCR primer/probe design tool and do not request unrelated credentials or install arbitrary remote code.

Purpose & Capability

The name/description (RT-qPCR primer and TaqMan probe design, homology mapping, high-GC optimization) match the included script and examples. Required resources (NCBI efetch calls, optional local FASTA input) are appropriate for the stated functionality; there are no unrelated environment variables, binaries, or external services requested.

Instruction Scope

SKILL.md instructs the agent to fetch sequences by accession or read a provided FASTA and run scripts/design_qpcr_assay.py with arguments (target, homolog, offtarget). The script performs only those actions: HTTP GETs to NCBI eutils and optional local FASTA file reads. There are no instructions to access unrelated system files, other credentials, or to transmit data to unknown endpoints.

Install Mechanism

This is instruction-only (no install spec). A requirements.txt lists 'requests' but no automated installer is provided; the agent or user will need Python + requests to run the script. This is a minor usability inconsistency, not a security concern, but the script will perform network calls when executed.

Credentials

The skill requests no environment variables, credentials, or config paths. Network access is only to NCBI eutils (expected). The ability to read a user-supplied FASTA file is necessary for local sequence inputs; it does not read arbitrary system config by default.

Persistence & Privilege

The skill is not always-enabled and is user-invocable; it does not request persistent privileges or modify other skills. Autonomous invocation is allowed by default but is not combined with broad credential access or other red flags.

Assessment

This skill appears coherent with its stated purpose. Before installing or running: 1) Review the Python script if you have concerns—it's included in the package and will make outbound HTTPS requests to NCBI (efetch) to retrieve sequences/annotations. 2) Running the script requires Python and the 'requests' package (requirements.txt present but no installer provided). 3) If you pass a local FASTA path, the script will read that file—don't point it at sensitive system files. 4) The sequence parsing and Tm calculations are simplified; validate any critical assay designs with your standard lab QC and established tools. 5) Because the source is unknown, consider running the script in a sandboxed environment or reviewing code before executing in production.

RT-qPCR Primer Design (Bio-chat Series)

This skill provides a professional-grade workflow for designing high-specificity RT-qPCR primers and probes, optimized for complex biological samples.

Key Capabilities

1. Homology-based Junction Mapping: Automatically maps exon-exon junctions from a well-annotated ortholog (e.g., Human NM_) to a predicted target transcript (e.g., Vero XM_) to ensure gDNA-safe design even when annotations are missing.
2. High-GC Template Optimization: Specialized algorithms for templates with >70% GC content (e.g., Pseudorabies virus), automatically adjusting primer length and Tm weight to ensure specificity.
3. SYBR & TaqMan Support: Designs optimized primer pairs for SYBR Green and triplex/probe sets for TaqMan assays with precise Tm matching.

Workflow

1. Target Identification: Provide a NCBI Accession (NM_/XM_) or a local FASTA sequence.
2. Homology Mapping (Optional): For predicted monkey transcripts, provide a Human ortholog Accession to activate "Hardcore Mode" for exon junction mapping.
3. Automated Design: Execute scripts/design_qpcr_assay.py with specific parameters:
   • --target: Target accession
   • --homolog: Human ortholog for mapping
   • --offtarget: Accession for 3' specificity check
4. Validation: Review the top-scoring primer/probe sets based on Tm, GC, 3' quality, and secondary structure.

Technical Standards

• Tm Calculation: Nearest-Neighbor (NN) model.
• gDNA Defense: Primers must span or flank exon-exon junctions.
• 3' Quality: Strict control of 3' GC-clamp and mismatch prevention.

Designed by ZJU PhD @ Bio-chat Community