RT-qPCR Primer Designer & Delivery Skill

Overview

This skill provides an end-to-end automated workflow to design high-specificity, thermodynamically verified primers for Reverse Transcription Quantitative PCR (RT-qPCR, SYBR Green I method) and directly outputs publication/lab-ready formatted Excel delivery workbooks (.xlsx).

Key capabilities:

• Sequence Retrieval: Automatically fetches mRNA sequence, parses CDS coordinates, UTRs, and gene annotations from NCBI GenBank accessions.
• Thermodynamic Primer Design: Leverages Primer3 to design primers targeting the CDS region with optimal amplicon sizes (80–150 bp), matching melting temperatures ($T_m \approx 60\ ^\circ\text{C}$), balanced GC content (40%–60%), and 3' GC clamps.
• Secondary Structure Filtering: Rigorously eliminates primers with stable hairpins ($T_m \ge 45\ ^\circ\text{C}$), self-dimers, or cross-dimers.
• Dual Specificity Verification: Scans 3' 10-mer seed binding sites across the target transcript, with NCBI BLAST genome/transcriptome validation and graceful offline fallback.
• Formatted Excel Delivery: Automatically produces a 3-sheet styled workbook containing:
  1. qPCR引物信息表: Ranked primer pairs, sequences, positions, amplicon length, GC%, and full amplicon sequences.
  2. 目标基因与序列详情: Full gene metadata, CDS coordinates, and nucleotide sequences.
  3. qPCR实验参考指南: 20 μL reaction master mix table, standard 2-step thermal cycling protocol, and quality control guidelines (DNase I treatment, NTC controls, reference genes).

Dependencies

• ncbi-sequence-fetch: Used for fetching NCBI sequence records and annotations.
• Python Libraries:
  • primer3-py: Thermodynamic nearest-neighbor calculations ($T_m$, hairpin, dimers).
  • openpyxl: Excel workbook formatting and styling.
  • biopython: Sequence parsing and BLAST query handling.

Quick Start

1. One-Stop End-to-End Pipeline

Given an NCBI accession number (e.g. OR123894.1):

python scripts/qpcr_designer.py pipeline \
  --accession OR123894.1 \
  --output "IFN_gamma_qPCR_primers.xlsx"

2. From Local FASTA File with Specified CDS

python scripts/qpcr_designer.py pipeline \
  --fasta "gene.fasta" \
  --cds-start 166 \
  --cds-end 771 \
  --organism "Nibea albiflora" \
  --product "IFN-gamma" \
  --output "gene_qpcr_primers.xlsx"

Utility Scripts (scripts/qpcr_designer.py)

The helper CLI supports both individual modular subcommands and the complete pipeline:

1. fetch

Fetches GenBank record by accession and saves structured metadata to JSON:

python scripts/qpcr_designer.py fetch \
  --accession OR123894.1 \
  -o "gene_meta.json"

2. design

Designs primer pairs within the CDS with specified amplicon length and $T_m$:

python scripts/qpcr_designer.py design \
  --input "gene_meta.json" \
  --min-prod 80 \
  --max-prod 150 \
  --opt-tm 60.0 \
  -o "candidates.json"

3. specificity

Performs transcript-level off-target scanning and adds specificity annotations:

python scripts/qpcr_designer.py specificity \
  --input "candidates.json" \
  -o "verified_candidates.json"

4. export-excel

Renders the verified primer data into a styled 3-sheet Excel delivery file:

python scripts/qpcr_designer.py export-excel \
  --input "verified_candidates.json" \
  -o "qPCR_Primers_Report.xlsx"

Workflow Guide

When the user asks to design qPCR primers:

1. Identify the Input:
   • GenBank accession / NCBI link $\rightarrow$ Call fetch or pipeline --accession.
   • FASTA sequence or raw text $\rightarrow$ Save to .fasta and specify --cds-start and --cds-end.
2. Execute Design & Specificity Verification:
   • Run pipeline with default 80–150 bp amplicon range and 60 °C annealing temperature.
3. Present Results in Chat:
   • Highlight the Top Recommended Pair and 1–2 Alternative Pairs with $T_m$, GC%, amplicon length, and amplicon sequence.
   • Summarize the BLAST/transcript specificity findings.
4. Deliver the Excel File:
   • Provide a clickable markdown file link to the generated .xlsx report.

Rate Limiting & Error Handling

• NCBI API Requests: Standard NCBI rate limit is 3 requests/second (or 10 req/s with NCBI_API_KEY).
• BLAST / Network Fallback: If NCBI BLAST times out or the target species has incomplete genome assembly in NCBI, the pipeline automatically falls back to transcript-level seed scan + closely related species homologous verification, surfacing a clear notice in the output.

Common Mistakes to Avoid

• Designing outside the CDS: Primers in long UTR regions may amplify non-coding splice variants. Always locate primers inside the coding region (CDS) unless specifically targeting UTR.
• Amplicon too long (>200 bp): Standard fast qPCR cycles (20–30 s extension) require short amplicons (80–150 bp). Longer amplicons lead to lower amplification efficiency.
• Ignoring gDNA Contamination: Always advise the user to treat RNA with DNase I or use gDNA-removing reverse transcriptase kits.